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研究生:柯世傑
研究生(外文):Shih-Ghieh Ke
論文名稱:利用分子生物方法進行樣本中硝化菌與硝化速率之估算
論文名稱(外文):Using molecular biology method to calculate the numbers of nitrifying bacteria and their nitrification rates in different samples
指導教授:趙 維 良
指導教授(外文):Wei-Liang Chao
學位類別:碩士
校院名稱:東吳大學
系所名稱:微生物學系
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2006
畢業學年度:93
語文別:中文
論文頁數:80
中文關鍵詞:硝化菌硝化速率原位螢光雜合法流式細胞儀
外文關鍵詞:Flow Cytometryfluorescent in situ hybridizationnitrifying bacterianitrification rates
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硝化作用為整個氮循環的必須步驟之一,這一連串的反應主要由自營性的硝化菌負責,因此,自營性硝化菌在氮循環中扮演著關鍵的角色。但由於硝化菌生長緩慢,且在培養過程中其自身代謝產物會降低生存環境之pH值,進而抑制自身生長。因自營菌生長速度緩慢,造成遭受其他微生物污染的機率上升,所以更增加了硝化菌在培養及分離上的困難,以及計數上的限制,使得現今環境中硝化菌的實際數量及其計數方式仍存有許多爭議。
基於上述原因,本研究希望以分子生物學的方法快速且精確的計數樣本中硝化菌的數量,所採用的方式為原位螢光雜合法 (FISH; Fluorescent In Situ Hybridization),針對銨氧化菌與亞硝酸氧化菌的16S rDNA設計一段專一性的探針。根據基因庫中硝化菌的序列,以GCG SeqWeb 軟體進行序列排序,比對後選取了對於銨氧化菌或亞硝酸氧化菌,具專一性的探針Nso1225、Nit3,並藉由聚合脢連鎖反應的適用性分析,確認其專一性;各探針雜合過程中的最適條件分別為探針Eub338:0%formamide、0.9M NaCl、探針Nso1225:35%formamide、0.08MNaCl、探針Nit3:40% formamide、0.056M NaCl。
本研究亦希望觀察到銨氧化菌菌數與銨氧化速率之間的關係,在純菌實驗中,銨氧化菌的Generation Time大約為9小時,亞硝酸氧化菌Generation Time大約12小時。而銨氧化菌的銨氧化速率為2.58×10-7μmol/cell•h,亞硝酸氧化菌的亞硝酸氧化速率為6.13×10-8 μmol/cell•h在土壤中每一個銨氧化菌的銨氧化速率為1.69×10-7 μmol/cell•h,亞硝酸氧化菌的亞硝酸氧化速率為4.17×10-8μmol/cell•h,以流式細胞儀分析土壤中銨氧化菌與亞硝酸氧化菌的成比例分別14.57%、12.65%,因此利用FISH結合流式細胞儀的方法可以較精確的進行樣本中硝化菌與硝化速率之估算。
Nitrification is one of the most important step is the nitrogen cycle, this series of reactions are responsible for chemoautotrophic nitrobacteria, therefore the chemoautotrophic nitrobacteria play an important role in nitrogen cycle. Nitrobacteria grow in a slow rate, and during their growth, they produce some acidic and toxic metabolites and that will inhibit their growth too. Because of the slow growth rate, that increases the contaminant opportunity by other micro organisms, for these reason it risen the difficulty of culturing and purifying the nitrobacteria, and limit the counting method.
According to those limitations, this study would like to use the molecular biology method to enumerate the nitrobacteria in the sample fast and precisely. We use fluorescent in situ hybridization method (FISH). First, we choose ammonia- oxidizing bacteria and nitrite- oxidizing bacteria 16S rDNA from the gene bank, then alignment by GCG SeqWeb software, to find out the the genus specific region sequences to be the FISH probe Nso1225, and Nit3. And to make sure the specificity of the probe, we use the probe as one side primer to do the PCR test. The result shows no cross reaction with other species of bacteria.
The FISH result shows that the optima conditions of different probes are Eub338: 0% formamide, 0.9M NaCl, Nso1225: 35% formamide, 0.08MNaCl, Nit3: 40% formamide, 0.056M NaCl.
Our study detects the ammonia- oxidation rate and the nitrite- oxidation rate of the sample, to find out the linkage of nitrification rate and the numbers of the ammonia- oxidizing bacteria and the nitrite- oxidizing bacteria. According to the results, the generation time of the ammonia- oxidizing bacteria in the medium are 9 hours, and the generation time of the nitrite- oxidizing bacteria in the medium is 12 hours. The ammonia-oxidation rate of the ammonia- oxidizers is 2.58 × 10-7 μmol/cell.h, and the nitrite-oxidation rate of the nitrite-oxidizers is 6.13 × 10-8 μmol/cell.h.
We also applied this experiment in soil sample, the ammonia- oxidation rate in the soil is 1.69 × 10-7 μmol/cell.h, and the nitrite-oxidation rate is 4.17 × 10-8 μmol/cell.h. The amount of ammonium-oxidizers and nitrite-oxidizers in the soil are counted by Flow Cytometry after hybridized by specific probes. The ratio of ammonium -oxidizers and nitrite-oxidizers are 14.57% and 12.65%.
According to our results, use both FISH and Flow Cytometry method to calculate the numbers of nitrifying bacteria and their nitrification rates in different samples more precisely.
目 錄
目錄………………………………………………….…………………………………i
表目錄…………………………………………………………………………………iv
圖目錄…………………………………………………………………………………v
中文摘要………………………………………………………………………………vii
英文摘要………………………………………………………………………………ix
壹、前言……………………………………………………………………………… 1
貳、實驗目的………………………………………………………………………… 9
參、材料方法………………………………………………………………………… 10
一、 硝化菌的培養、篩選及分離………………………………………………….10
二、 硝化菌染色體DNA的萃取…………………………………………………..11
三、探針的設計…………………………………………………………………….13
四、利用PCR進行探針適用性的測試…………………………………………….13
五、原位螢光雜合分析…………………………………………………………….14
1.菌體固定……………………………………………………………………..14
2.菌體脫水……………………………………………………………………..15
3.菌體雜合……………………………………………………………………..15
六、螢光顯微照相………………………………………………………………….16
七、流式細胞儀分析…………………………………………………………………16
八、土壤菌數回收率測試……………………………………………………………16
九、短期硝化活性之測定……………………………………………………………17
1.各種無機氮的測試…………………………………………………………….17
2.硝化菌短期硝化活性的測試………………………………………………….18
3.土壤短期硝化活性的測試…………………………………………………….19
十、硝化菌生長速率分析……………………………………………………………19
肆、結果…………………………………………………………………………………21
一、硝化菌的培養、篩選及分離……………………………………………………21
二、探針的設計………………………………………………………………………21
三、利用PCR進行探針適用性的測試………………………………………………22
四、硝化菌螢光雜合條件測試………………………………………………………23
五、硝化菌短期硝化活性之測定……………………………………………………24
六、土壤回收率測試…………………………………………………………………26
七、流式細胞儀分析…………………………………………………………………26
八、FISH與流式細胞儀分析比較…………………………………………………..27
九、硝化菌生長速率…………………………………………………………………27
伍、討論…………………………………………………………………………………29
一、硝化菌的培養、篩選及分離……………………………………………………29
二、探針的設計與適用性……………………………………………………. ……..29
三、螢光雜合條件測試………………………………………………………………30
四、硝化菌短期硝化活性之測定……………………………………………………31
五、土壤回收率測試…………………………………………………………………32
六、流式細胞儀分析…………………………………………………………………33
七、硝化菌生長速率…………………………………………………………………34
參考文獻…………………………………………………………………………………35
圖……………………………………………………………………………………..…..42
表……………………………………………………………………………..…………..63
附錄………………………………………………………………………………………67

表 目 錄
表一、銨氧化菌培養基之成分…………………………………..…………………………63
表二、本研究所使用探針與引子序列…………………….………………….............64
表三、以FISF方法所測的的菌數與時間關係表…………………………………………65
表四、亞硝酸氧化菌培養基之成分………………………………………………………66

圖 目 錄
圖一、探針EUB338序列比對分析圖………………………………………….………… 42
圖二、探針Nso1225序列比對分析圖……………………………………………………43
圖三、探針Nit3序列比對分析圖…………………………………………………………..44
圖四、銨氧化菌探針適用性測試,以不同引子進行PCR結果…………………………..45
圖五、亞硝酸氧化菌探針適用性測試,以不同引子進行PCR結果……………………46
圖六、以探針Eub338對E. coli進行雜合反應………………………………………….47
圖七、以探針Nso1225對銨氧化菌進行雜合反應………………………………………48
圖八、以探針Nit3對亞硝酸氧化菌進行雜合反應………………………………………..49
圖九、銨態氮濃度與波長636nm吸光值之關係圖………………………………………..50
圖十、亞硝酸態氮濃度與波長540nm吸光值之關係圖…………………………………..51
圖十一、硝酸態氮濃度與波長410nm吸光值之關係圖…………………………………..52
圖十二、硝化菌在銨氧化菌培養基中銨態氮與亞硝酸態氮的關係圖…………………53
圖十三、硝化菌在亞硝酸氧化菌培養基中亞硝酸態氮與硝酸態的關係圖……………54
圖十四、硝化菌在土壤中銨態氮與亞硝酸態氮的關係圖………………………………55
圖十五、硝化菌在土壤中亞硝酸態氮與硝酸態的關係圖……………………………….56
圖十六、Micrococcus lateus土壤回收率分析……………………………………………57
圖十七、流式細胞儀分析培養基中銨氧化菌比例………………………………………58
圖十八、流式細胞儀分析培養基中亞硝酸氧化菌比例…………………………………59
圖十九、流式細胞儀分析土壤中銨氧化菌比例…………………………………………60
圖二十、流式細胞儀分析土壤中亞硝酸氧化菌比例……………………………………61
圖二十一、土壤樣本中銨氧化菌與亞硝酸氧化菌的組成比例…………………………62
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