阿維菌素降解菌株的分離、鑒定及生物降解途徑研究
發(fā)布時(shí)間：2020-10-30 05:43
阿維菌素是除蟲(chóng)菌素B1a的商業(yè)化產(chǎn)品。除蟲(chóng)菌素B1a廣泛用于防治各種動(dòng)物體內(nèi)及體表寄生蟲(chóng),農(nóng)作物、觀賞性植物、蔬菜、水果等的害蟲(chóng),以及防治火蟻。阿維菌素殘留已經(jīng)造成了嚴(yán)重的環(huán)境污染。一方面阿維菌素對(duì)土壤或糞便中的無(wú)脊椎動(dòng)物有毒害作用,其使用可能導(dǎo)致當(dāng)?shù)厣鷳B(tài)系統(tǒng)失衡,更嚴(yán)重的是,阿維菌素殘留很容易隨雨水等從施用地點(diǎn)進(jìn)入附近的水域。即使在很低的濃度下,阿維菌素對(duì)水生生物都有很高的毒性。因此,開(kāi)發(fā)一種修復(fù)技術(shù)除去環(huán)境中的阿維菌素殘留顯得十分必要。由于具有高效性和低成本等優(yōu)點(diǎn),微生物修復(fù)技術(shù)成為去除環(huán)境中污染物的理想選擇之一 通過(guò)連續(xù)富集培養(yǎng),我們從長(zhǎng)年受阿維菌素污染的橘園土壤中分離到一株新的阿維菌素降解菌GB-01.GB-01能夠利用阿維菌素作為唯一碳源在礦物鹽培養(yǎng)基中生長(zhǎng)。使用光學(xué)顯微鏡和透射電子顯微鏡(TEM)觀察了GB-01的菌體顯微形態(tài)。參照伯杰細(xì)菌鑒定手冊(cè),我們進(jìn)行了一系列的生化試驗(yàn)對(duì)該菌進(jìn)行鑒定。GB-01是一株革蘭氏陽(yáng)性菌,可形成直徑約0.6-1毫米的表面光滑菌落。GB-01細(xì)胞呈直桿狀,兩端鈍圓,寬0.4-0.7μm,長(zhǎng)1.2-1.7gmm,有時(shí)兩個(gè)細(xì)胞會(huì)形成首尾相連的短鏈。根據(jù)形態(tài)學(xué)和生化特征,GB-01被初步鑒定為伯克霍爾德菌(Burkholderia)。然后我們使用了多種分類學(xué)方法來(lái)鑒定GB-01。使用Biolog GN2 MicroPlating、API 20NE、ID 32 GN.和API 50 CH多種試劑盒鑒定其生理生化特征。我們也研究了GB-01的化學(xué)分類學(xué)特征如G+C含量和細(xì)胞脂肪酸構(gòu)成。PCR擴(kuò)增GB-01的16S rRNA和recA基因序列并進(jìn)行測(cè)序,從Genbank (?)數(shù)據(jù)庫(kù)中下載相關(guān)序列,并用Clustal X 1.8.3(默認(rèn)參數(shù))進(jìn)行序列比對(duì)。使用MEGA 4.0分析系統(tǒng)發(fā)育關(guān)系,用Kimura 2參數(shù)模型計(jì)算遺傳距離,然后用neighbor-joining方法構(gòu)建系統(tǒng)發(fā)育樹(shù)。通過(guò)和親緣關(guān)系較近種的DNA-DNA雙雜交,我們獲得了另一關(guān)鍵數(shù)據(jù)。多相分類分析表明菌株GB-01屬于洋蔥伯克霍爾德菌群(Bcc),是一種非典型的Burkholderia diffusa菌株。 菌株GB-01的最適生長(zhǎng)條件是pH 7.0,溫度30℃。我們分別在液體培養(yǎng)基和土壤中進(jìn)行了生物降解試驗(yàn)。向液體MSM培養(yǎng)基中添加50 mg L-1或100 mg L-1的阿維菌素作為唯一碳源,30或36h后GB-01能夠利用多于90%的阿維菌素。在所有的搖瓶培養(yǎng)試驗(yàn)中,種子液菌體密度均為1.50D(OD600),接種量均為2%(v/v)。在肉湯培養(yǎng)基中,最適的降解條件也是溫度30℃,pH 7。然而,在溫度35℃,pH 8培養(yǎng)條件下和最適條件下的降解效果沒(méi)有明顯區(qū)別。當(dāng)pH低于5或高于9,溫度低于10℃或高于45℃時(shí),GB-01的降解能力迅速下降。MSM培養(yǎng)基中添加阿維菌素的初始濃度會(huì)顯著地影響GB-01的降解能力。當(dāng)阿維菌素濃度高于100 mg L-1時(shí),降解周期變長(zhǎng)。GB-01不能降解初始濃度為200 mg L-1的阿維菌素。另外,種子液細(xì)胞培養(yǎng)到對(duì)數(shù)生長(zhǎng)期時(shí)添加2mg L-1的阿維菌素誘導(dǎo)能夠顯著的提高GB-01的降解效率。 盆缽試驗(yàn)中,GB-01能夠有效降解土壤中的阿維菌素殘留。這些表明GB-01很有潛力作為生物修復(fù)菌株用于大田修復(fù)。GB-01降解土壤中阿維菌素殘留的最適溫度為30-35℃,中性至偏堿性的土壤(如pH 7-8)能夠顯著的提高降解效果。這表明GB-01可以用于不同的土壤。當(dāng)每克干土接種108cfu(菌落形成單位)時(shí),降解效果最好,在最適溫度和pH條件下,GB-01能夠有效降解阿維菌素(50 mg Kg-1)。這表明即使在阿維菌素過(guò)度使用或不慎溢灑的區(qū)域,GB-01也能夠有效去除阿維菌素殘留。接種頻率也對(duì)降解效果有明顯的影響。與單次接種相比,定期連續(xù)多次接種的降解速度更快。接種前的誘導(dǎo)也會(huì)提高降解速率。淹水條件對(duì)菌株GB-01的生物降解能力并沒(méi)有顯著影響。 我們分離并鑒定了培養(yǎng)基萃取液中的阿維菌素降解產(chǎn)物。通過(guò)高效液相色譜串聯(lián)質(zhì)譜(HPLC-MS/MS),我們檢測(cè)到GB-01降解阿維菌素產(chǎn)生的兩個(gè)新代謝產(chǎn)物。并根據(jù)其質(zhì)譜數(shù)據(jù)和特征碎片類型對(duì)這兩個(gè)產(chǎn)物進(jìn)行鑒定�；谶@些發(fā)現(xiàn),我們提出了一個(gè)阿維菌素生物降解的部分途徑。 總之,我們從多個(gè)角度研究了菌株GB-01對(duì)阿維菌素的降解作用。GB-01可以降解高濃度的阿維菌素,具有獨(dú)特有效的降解途徑,并且可以去除污染土壤中的阿維菌素殘留。據(jù)我們所知,GB-01是第一次報(bào)道的能夠在有氧條件下降解阿維菌素且分離于土壤土著微生物群體的伯克霍爾德菌。結(jié)果表明Burkholderia diffusa GB-01菌株可以有效用于阿維菌素污染土壤的生物修復(fù)。除此之外,這是對(duì)分離于環(huán)境中的Burkholderia diffusa菌株生物修復(fù)潛力的第一次研究。
【學(xué)位單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位年份】：2010
【中圖分類】：X172
【文章目錄】：
摘要
ABSTRACT
LIST OF ABBREVIATIONS
LIST OF TABLES
LIST OF FIGURES
CHAPTER 1 INTRODUCTION
CHAPTER 2 REVIEW OF LITERATURE
1. AVERMECTINS
2. ABAMECTIN
3. GENERAL PROPERTIES
4. PHYSICAL/CHEMICAL PROPERTIES
5. MODE OF ACTION
6. METABOLISM
7. TOXICITY
8. ENVIRONMENTAL FATE
8.1. Concentrations and persistence in environmental compartments
8.2. Impacts on terrestrial fauna
8.2.1. Lethal impacts
8.2.2. Sublethal impacts
8.3. Impacts on aquatic fauna
8.3.1. Impacts on target aquatic animals
8.3.2. Impacts on non-target marine organisms
8.3.3. Impacts on non-target freshwater organisms
9. BIODEGRADATION/BIOREMEDIATION
References
CHAPTER 3 ISOLATION AND CHARACTERIZATION OF BACTERIAL STRAIN GB-01
Abstract
1. INTRODUCTION
2. MATERIALS AND METHODS
2.1 Chemicals
2.2 Culture media
2.2.1 Washing and sterilization of glassware
2.2.2 Medium preparation
2.3. Enrichment of the abamectin-degrading consortium
2.4 Isolation of pure culture
2.5 Maintenance of bacterial culture
2.6 Identification and characterization of strain GB-01
2.6.1 Morphological characterization
2.6.1.1 Transmission electron microscopy
2.6.1.2 Smear preparation and Gram staining
2.6.1.3 Cultural characterization
2.6.2 Physiological and biochemical characterization
2.6.2.1 Fermentation of sugars
2.6.2.2 Methyl red and Voges Praskaeur test
2.6.2.3 Indole test
2.6.2.4 Citrate utilization test
2.6.2.5 Nitrate reduction test
2.6.2.6 Catalase test
2.6.2.7 Liquefication of gelatin
2.7 Biodegradation assay of abamectin by strain GB-01
2.7.1 Inoculum preparation
2.7.2 Construction of reactors
2.7.3 Effects of Temperature
2.7.4 Effects of pH
2.7.5 Effects of initial concentration
2.7.6 Biodegradation kinetics
2.8 Analytical techniques
2.8.1 Extraction of abamectin for HPLC
2.8.2 High pressure liquid chromatography （HPLC）
2.8.3 Calibration of HPLC instrument
3. RESULTS AND DISCUSSION
3.1 Degradability of abamectin through enrichment culture
3.2 Isolation and characterization of the strain GB-01
3.3 Biodegradation of abamectin by strain GB-01 in cell culture
3.4 Degradation at different temperatures
3.5 Degradation at different pH
3.6 Degradation at different initial concentrations of abamectin
4. CONCLUSION
References
CHAPTER 4 TAXONOMIC ANALYSIS OF STRAIN GB-01
Abstract
1. INTRODUCTION
1.1 History and structure of the Burkholderia genus
1.2 Natural diversity and potential application of Burkholderia cepacia complex species
1.3 Identification and Classification of Bcc
1.4 Research aim
2. MATERIALS AND METHODS
2.1 Bacterial strain and culture conditions
2.2 Genomic DNA extraction
2.3 DNA quantification and purity check
2.4 Polymerase chain reaction （PCR）
2.5 Agarose gel electrophoresis
2.6 DNA （PCR product） purification from agarose gel
2.7 Cloning of PCR product
2.8 Transformation of E. coli
2.9 Plasmid purification from E. coli
2.10 Restriction endonuclease digestion
2.11 Cycle sequencing of DNA
2.12 Molecular sequence analysis
2.13 Biochemical and substrate utilization test
2.13.1 API 20 NE
2.13.2 ID 32 GN
2.13.3 API 50 CH
2.13.4 Biolog GN2 MicroPlate
2.14 Whole cell fatty acid profiling
2.15 Whole genome G+C mol% determination
2.16 Whole genome DNA-DNA hybridization
3. RESULTS AND DISCUSSION
3.1 Biochemical and substrate utilization characteristics of strain GB-01
3.2 Whole cell fatty acid profile of strain GB-01
3.3 G+C mol% of Strain GB-01
3.4 Phylogenetic analysis of 16S rRNA gene
3.5 Amplification of recA gene fragments from strain GB-01
3.6 Phylogenetic analysis of recA gene
3.7 Whole genome DNA-DNA hybridization
4. CONCLUSION
References
CHAPTER 5 BIODEGRADATION PATHWAY OF ABAMECTIN BY STRAIN GB-01
Abstract
1. INTRODUCTION
1.1 High performance liquid chromatography （HPLC）
1.2 Mass spectrometry
1.2.1 Triple quadrupole mass spectrometry
1.3 Electrospray Ionization （ESI）
2. MATERIALS AND METHODS
2.1 Chemicals, bacterium and culture media
2.2 Instrumentation
2.3 Biodegradation experimental system
2.4 Sample preparation
3. RESULTS AND DISCUSSION
3.1 Degradation products
3.2 Structural elucidation of the metabolites
4. CONCLUSION
References
CHAPTER 6 EVALUATION OF BIOREMEDIATION POTENTIAL OF STRAIN GB-01 IN SOIL MICROCOSMS
Abstract
1. INTRODUCTION
2. MATERIALS AND METHODS
2.1 Chemicals, organism and culture conditions
2.2 Effect of different factors on the growth of strain GB-01
2.3 Induction studies
2.4 Preparation of the inoculum and soil microcosms
2.5 Study of different factors affecting the abamectin degradation in soil microcosms
2.6 Extraction and HPLC analysis
2.7 Reproducibility
3. RESULTS AND DISCUSSION
3.1 Growth characterization of strain GB-01 for inoculum preparation
3.2 Effects of substrate concentration
3.3 Effects of pH and temperature
3.4 Effects of inoculum size, inoculation frequency and flooded conditions
4. CONCLUSION
References
SUMMARY AND CONCLUSIONS
SIGNIFICANCE OF THE RESEARCH
FUTURE PERSPECTIVES
PUBLICATIONS
APPENDICES
ACKNOWLEDGMENT
本文編號(hào)：2862073
【學(xué)位單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位年份】：2010
【中圖分類】：X172
【文章目錄】：
摘要
ABSTRACT
LIST OF ABBREVIATIONS
LIST OF TABLES
LIST OF FIGURES
CHAPTER 1 INTRODUCTION
CHAPTER 2 REVIEW OF LITERATURE
1. AVERMECTINS
2. ABAMECTIN
3. GENERAL PROPERTIES
4. PHYSICAL/CHEMICAL PROPERTIES
5. MODE OF ACTION
6. METABOLISM
7. TOXICITY
8. ENVIRONMENTAL FATE
8.1. Concentrations and persistence in environmental compartments
8.2. Impacts on terrestrial fauna
8.2.1. Lethal impacts
8.2.2. Sublethal impacts
8.3. Impacts on aquatic fauna
8.3.1. Impacts on target aquatic animals
8.3.2. Impacts on non-target marine organisms
8.3.3. Impacts on non-target freshwater organisms
9. BIODEGRADATION/BIOREMEDIATION
References
CHAPTER 3 ISOLATION AND CHARACTERIZATION OF BACTERIAL STRAIN GB-01
Abstract
1. INTRODUCTION
2. MATERIALS AND METHODS
2.1 Chemicals
2.2 Culture media
2.2.1 Washing and sterilization of glassware
2.2.2 Medium preparation
2.3. Enrichment of the abamectin-degrading consortium
2.4 Isolation of pure culture
2.5 Maintenance of bacterial culture
2.6 Identification and characterization of strain GB-01
2.6.1 Morphological characterization
2.6.1.1 Transmission electron microscopy
2.6.1.2 Smear preparation and Gram staining
2.6.1.3 Cultural characterization
2.6.2 Physiological and biochemical characterization
2.6.2.1 Fermentation of sugars
2.6.2.2 Methyl red and Voges Praskaeur test
2.6.2.3 Indole test
2.6.2.4 Citrate utilization test
2.6.2.5 Nitrate reduction test
2.6.2.6 Catalase test
2.6.2.7 Liquefication of gelatin
2.7 Biodegradation assay of abamectin by strain GB-01
2.7.1 Inoculum preparation
2.7.2 Construction of reactors
2.7.3 Effects of Temperature
2.7.4 Effects of pH
2.7.5 Effects of initial concentration
2.7.6 Biodegradation kinetics
2.8 Analytical techniques
2.8.1 Extraction of abamectin for HPLC
2.8.2 High pressure liquid chromatography （HPLC）
2.8.3 Calibration of HPLC instrument
3. RESULTS AND DISCUSSION
3.1 Degradability of abamectin through enrichment culture
3.2 Isolation and characterization of the strain GB-01
3.3 Biodegradation of abamectin by strain GB-01 in cell culture
3.4 Degradation at different temperatures
3.5 Degradation at different pH
3.6 Degradation at different initial concentrations of abamectin
4. CONCLUSION
References
CHAPTER 4 TAXONOMIC ANALYSIS OF STRAIN GB-01
Abstract
1. INTRODUCTION
1.1 History and structure of the Burkholderia genus
1.2 Natural diversity and potential application of Burkholderia cepacia complex species
1.3 Identification and Classification of Bcc
1.4 Research aim
2. MATERIALS AND METHODS
2.1 Bacterial strain and culture conditions
2.2 Genomic DNA extraction
2.3 DNA quantification and purity check
2.4 Polymerase chain reaction （PCR）
2.5 Agarose gel electrophoresis
2.6 DNA （PCR product） purification from agarose gel
2.7 Cloning of PCR product
2.8 Transformation of E. coli
2.9 Plasmid purification from E. coli
2.10 Restriction endonuclease digestion
2.11 Cycle sequencing of DNA
2.12 Molecular sequence analysis
2.13 Biochemical and substrate utilization test
2.13.1 API 20 NE
2.13.2 ID 32 GN
2.13.3 API 50 CH
2.13.4 Biolog GN2 MicroPlate
2.14 Whole cell fatty acid profiling
2.15 Whole genome G+C mol% determination
2.16 Whole genome DNA-DNA hybridization
3. RESULTS AND DISCUSSION
3.1 Biochemical and substrate utilization characteristics of strain GB-01
3.2 Whole cell fatty acid profile of strain GB-01
3.3 G+C mol% of Strain GB-01
3.4 Phylogenetic analysis of 16S rRNA gene
3.5 Amplification of recA gene fragments from strain GB-01
3.6 Phylogenetic analysis of recA gene
3.7 Whole genome DNA-DNA hybridization
4. CONCLUSION
References
CHAPTER 5 BIODEGRADATION PATHWAY OF ABAMECTIN BY STRAIN GB-01
Abstract
1. INTRODUCTION
1.1 High performance liquid chromatography （HPLC）
1.2 Mass spectrometry
1.2.1 Triple quadrupole mass spectrometry
1.3 Electrospray Ionization （ESI）
2. MATERIALS AND METHODS
2.1 Chemicals, bacterium and culture media
2.2 Instrumentation
2.3 Biodegradation experimental system
2.4 Sample preparation
3. RESULTS AND DISCUSSION
3.1 Degradation products
3.2 Structural elucidation of the metabolites
4. CONCLUSION
References
CHAPTER 6 EVALUATION OF BIOREMEDIATION POTENTIAL OF STRAIN GB-01 IN SOIL MICROCOSMS
Abstract
1. INTRODUCTION
2. MATERIALS AND METHODS
2.1 Chemicals, organism and culture conditions
2.2 Effect of different factors on the growth of strain GB-01
2.3 Induction studies
2.4 Preparation of the inoculum and soil microcosms
2.5 Study of different factors affecting the abamectin degradation in soil microcosms
2.6 Extraction and HPLC analysis
2.7 Reproducibility
3. RESULTS AND DISCUSSION
3.1 Growth characterization of strain GB-01 for inoculum preparation
3.2 Effects of substrate concentration
3.3 Effects of pH and temperature
3.4 Effects of inoculum size, inoculation frequency and flooded conditions
4. CONCLUSION
References
SUMMARY AND CONCLUSIONS
SIGNIFICANCE OF THE RESEARCH
FUTURE PERSPECTIVES
PUBLICATIONS
APPENDICES
ACKNOWLEDGMENT
本文編號(hào)：2862073
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