阿维菌素降解菌株的分离、鉴定及生物降解途径研究
发布时间：2020-10-30 05:43
阿维菌素是除虫菌素B1a的商业化产品。除虫菌素B1a广泛用于防治各种动物体内及体表寄生虫,农作物、观赏性植物、蔬菜、水果等的害虫,以及防治火蚁。阿维菌素残留已经造成了严重的环境污染。一方面阿维菌素对土壤或粪便中的无脊椎动物有毒害作用,其使用可能导致当地生态系统失衡,更严重的是,阿维菌素残留很容易随雨水等从施用地点进入附近的水域。即使在很低的浓度下,阿维菌素对水生生物都有很高的毒性。因此,开发一种修复技术除去环境中的阿维菌素残留显得十分必要。由于具有高效性和低成本等优点,微生物修复技术成为去除环境中污染物的理想选择之一 通过连续富集培养,我们从长年受阿维菌素污染的橘园土壤中分离到一株新的阿维菌素降解菌GB-01.GB-01能够利用阿维菌素作为唯一碳源在矿物盐培养基中生长。使用光学显微镜和透射电子显微镜(TEM)观察了GB-01的菌体显微形态。参照伯杰细菌鉴定手册,我们进行了一系列的生化试验对该菌进行鉴定。GB-01是一株革兰氏阳性菌,可形成直径约0.6-1毫米的表面光滑菌落。GB-01细胞呈直杆状,两端钝圆,宽0.4-0.7μm,长1.2-1.7gmm,有时两个细胞会形成首尾相连的短链。根据形态学和生化特征,GB-01被初步鉴定为伯克霍尔德菌(Burkholderia)。然后我们使用了多种分类学方法来鉴定GB-01。使用Biolog GN2 MicroPlating、API 20NE、ID 32 GN.和API 50 CH多种试剂盒鉴定其生理生化特征。我们也研究了GB-01的化学分类学特征如G+C含量和细胞脂肪酸构成。PCR扩增GB-01的16S rRNA和recA基因序列并进行测序,从Genbank (?)数据库中下载相关序列,并用Clustal X 1.8.3(默认参数)进行序列比对。使用MEGA 4.0分析系统发育关系,用Kimura 2参数模型计算遗传距离,然后用neighbor-joining方法构建系统发育树。通过和亲缘关系较近种的DNA-DNA双杂交,我们获得了另一关键数据。多相分类分析表明菌株GB-01属于洋葱伯克霍尔德菌群(Bcc),是一种非典型的Burkholderia diffusa菌株。 菌株GB-01的最适生长条件是pH 7.0,温度30℃。我们分别在液体培养基和土壤中进行了生物降解试验。向液体MSM培养基中添加50 mg L-1或100 mg L-1的阿维菌素作为唯一碳源,30或36h后GB-01能够利用多于90%的阿维菌素。在所有的摇瓶培养试验中,种子液菌体密度均为1.50D(OD600),接种量均为2%(v/v)。在肉汤培养基中,最适的降解条件也是温度30℃,pH 7。然而,在温度35℃,pH 8培养条件下和最适条件下的降解效果没有明显区别。当pH低于5或高于9,温度低于10℃或高于45℃时,GB-01的降解能力迅速下降。MSM培养基中添加阿维菌素的初始浓度会显著地影响GB-01的降解能力。当阿维菌素浓度高于100 mg L-1时,降解周期变长。GB-01不能降解初始浓度为200 mg L-1的阿维菌素。另外,种子液细胞培养到对数生长期时添加2mg L-1的阿维菌素诱导能够显著的提高GB-01的降解效率。 盆钵试验中,GB-01能够有效降解土壤中的阿维菌素残留。这些表明GB-01很有潜力作为生物修复菌株用于大田修复。GB-01降解土壤中阿维菌素残留的最适温度为30-35℃,中性至偏碱性的土壤(如pH 7-8)能够显著的提高降解效果。这表明GB-01可以用于不同的土壤。当每克干土接种108cfu(菌落形成单位)时,降解效果最好,在最适温度和pH条件下,GB-01能够有效降解阿维菌素(50 mg Kg-1)。这表明即使在阿维菌素过度使用或不慎溢洒的区域,GB-01也能够有效去除阿维菌素残留。接种频率也对降解效果有明显的影响。与单次接种相比,定期连续多次接种的降解速度更快。接种前的诱导也会提高降解速率。淹水条件对菌株GB-01的生物降解能力并没有显著影响。 我们分离并鉴定了培养基萃取液中的阿维菌素降解产物。通过高效液相色谱串联质谱(HPLC-MS/MS),我们检测到GB-01降解阿维菌素产生的两个新代谢产物。并根据其质谱数据和特征碎片类型对这两个产物进行鉴定。基于这些发现,我们提出了一个阿维菌素生物降解的部分途径。 总之,我们从多个角度研究了菌株GB-01对阿维菌素的降解作用。GB-01可以降解高浓度的阿维菌素,具有独特有效的降解途径,并且可以去除污染土壤中的阿维菌素残留。据我们所知,GB-01是第一次报道的能够在有氧条件下降解阿维菌素且分离于土壤土著微生物群体的伯克霍尔德菌。结果表明Burkholderia diffusa GB-01菌株可以有效用于阿维菌素污染土壤的生物修复。除此之外,这是对分离于环境中的Burkholderia diffusa菌株生物修复潜力的第一次研究。
【学位单位】：南京农业大学
【学位级别】：博士
【学位年份】：2010
【中图分类】：X172
【文章目录】：
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
本文编号：2862073
【学位单位】：南京农业大学
【学位级别】：博士
【学位年份】：2010
【中图分类】：X172
【文章目录】：
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
本文编号：2862073
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