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研究生:李旻浥
研究生(外文):Min-Yi Li
論文名稱:柴油污染土壤風化過程中石油碳氫化合物功能性降解酶分佈特性分析
論文名稱(外文):Distribution Characteristics of Petroleum Hydrocarbon Functional Degradation Enzymes in Diesel-weathering Processes of Contaminated Soils
指導教授:田倩蓉田倩蓉引用關係
指導教授(外文):Dr. Chien-Jung Tien
學位類別:碩士
校院名稱:國立高雄師範大學
系所名稱:生物科技系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:175
中文關鍵詞:柴油土壤功能性降解酶風化
外文關鍵詞:Diesel fuelSoil function degradation enzymeWeathering
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土壤中碳氫化合物功能性降解酶在柴油污染土壤風化過程中的微生物降解作用扮演相當重要的角色。因此,本研究透過分析土壤中主要功能性降解酶基因表現在柴油污染土壤中之分佈與時間變化趨勢,以及分析各環境因子(如pH、含水率、有機碳、總氮與總磷含量)對其影響,來瞭解其在柴油風化過程之可能生物降解途徑。
在經過六個月的實驗期之後,以有添加泥炭土且未滅菌的實驗土樣的柴油降解率最優。有滅菌的組別較未滅菌組皆有較低之土壤總菌落數。而土壤的pH值自pH 7下降至pH 5左右,含水率自15%降至約5%,可能對部分微生物的生長產生影響。在未添加泥炭土的土樣中有機碳含量約為2%,有添加泥炭土的土樣約為4%。有添加泥炭土的土樣總氮含量皆比未添加泥炭土土樣高,南部土的總氮含量高於北部土;土壤總磷含量除了南部土初始月份以外,其餘皆低於500 μg/g dw;土壤碳磷比與氮磷比於北部土樣中皆隨時間增加而上升,南部土樣則僅在初始至第三月份均有上升,且其比值皆在實驗中期逐漸轉化為適合微生物生長之條件。
在土壤中功能性降解酶基因之定量結果發現,南部土出現的土壤功能性降解酶總數量、月份與mRNA表現值高於未污染的控制組(相對比值>1)的組別數皆較北部土多,並且添加泥炭土與否並不會直接影響到功能性降解酶mRNA表現量。在各個土壤功能性降解酶表現的部分,降解直鏈烷的alkB基因在初始到第四個月間表現;xylE基因分佈於不添加泥炭土的第四與第五月份;nahAc基因於初始至第六月份皆有出現,並且以未添加泥炭土的土樣有較多的mRNA表現比值高於1的狀況;bphA基因在未添加泥炭土的土樣中於第三個月後開始表現,在有添加泥炭土的土樣中則在第一個月開始有mRNA表現且分佈月份較多;bphA1除了初始月份以外皆有DNA出現,是否添加泥炭土對bphA1基因表現與否沒有明顯的影響;xylM基因僅在受台亞污染之南部土組別的初始與第二個月有表現;ntnM基因僅在兩種土樣的初始月份出現;tmoF基因在在未添加泥炭土的組別集中在前兩個月份,而有添加泥炭土的組別則自初始至第六個月皆有基因表現。整體而言,除了降解直鏈烷的alkB基因以外,其他降解支鏈與芳香族類的土壤功能性降解酶則較不受時間限制,顯示微生物會優先降解易分解的直鏈烷類,待直鏈烷類降解後陸續消耗較難被分解的環狀碳氫化合物。
土壤功能性降解酶與總石油碳氫化合物濃度、總菌落數以及土壤總磷含量呈顯著正相關性,顯示受到柴油污染的土壤中,具有足夠的碳源與磷源使具有功能性降解酶之微生物生長,促使土壤總菌落數增加。

Soil functional hydrocarbon-degrading enzymes play a relatively important role in diesel weathering processes. Thus, this study would like to analyze distribution and temporal change of gene expression of major functional degrading enzymes in diesel-contaminated soils, and determine effects of environmental factors (e.g. pH, water content, and concentration of organic carbon, total nitrogen and total phosphorous) in order to understand the possible biodegrading pathway in diesel weathering processes.
After the six-month experimental period, the experimental group of unsterilized diesel-contaminated soil with peat soil addition showed the highest diesel degradation rate among sixteen experimental groups. The sterilized experimental groups had lower number of bacterial colonies than the unsterilized ones. The soil pH decreased from pH 7 to pH 5 and the water content decreased from 15% to 5%, indicating the growth of some bacteria might be affected. The organic carbon content of soils without peat soil addition was about 2% and that of soils with peat soil addition was about 4%. The soils with peat soil addition contained higher total nitrogen than those without. The soils from the southern Taiwan contained higher total nitrogen than those from the northern Taiwan. The concentrations of total phosphorous were lower than 500 g/g dw, except for the soil from the southern Taiwan in the initial stage. The C/P and N/P increased with time in the soils from the northern Taiwan, but only shown for the soil from the southern Taiwan in the initial three-month stage. The C/P and N/P became suitable for growth of microorganisms in the later experimental period.
The quantitative results of gene expression of functional degrading enzymes showed that the soils from the southern Taiwan had higher gene expression of functional degrading enzymes (with relative ratio >1) than those from the northern Taiwan. The alkB for degrading n-alkane expressed in the initial four months. The xylE presented in the soils without peat soil addition in the 4th and 5th month. The nahAc was found in the six-month experimental period and expressed higher (with relative ration >1 for mRNA expression) in soils without peat soil addition. The bphA expressed after the 3rd month in soils without peat soil addition, but it expressed in the first month in the soil with peat soil addition. The bphA1 presented only in the later months. The xylM only found in the Formosa diesel-contaminated southern soils in the 1st and 2nd month. The ntnM only found in the two types of soils. The tmoF in soils without peat soil addition expressed in the initial two months, but that in soil with peat soil addition expressed in the six-month experimental period. To sum up, the alkB expressed in the initial months, but the other genes of functional degradation enzymes expressed during the six-month experimental period. It indicated that microorganisms would firstly degrade n-alkane, and then degrade aromatic hydrocarbons.
The gene expression of functional degrading enzymes was significantly positively correlated to concentrations of petroleum hydrocarbons and total phosphorous, and bacterial colonies. It indicated that the diesel-contaminated soil contained enough carbon and phosphorous to improve the growth of bacteria with functional degrading enzymes.
目錄
摘要 Ⅰ
Abstract Ⅱ
第一章 序論 1
1.1 研究動機 1
1.2 研究目的 3
第二章 文獻回顧 4
2.1 柴油基本性質與毒性 4
2.2 柴油洩漏與風化過程 5
2.2.1 柴油洩漏實例 5
2.2.2 柴油風化過程 6
2.3 柴油風化機制 7
2.3.1 揮發與溶解作用 8
2.3.2 生物降解作用 9
2.3.3 其他機制作用 9
2.4 微生物對柴油降解風化的影響 10
2.4.1 柴油之微生物降解路徑 10
2.4.2 影響柴油生物降解風化過程之相關因子介紹 27
2.5 即時聚合酶鏈鎖反應(Real-time polymerase chain reaction, Real-Time PCR) 28
2.5.1 Real-Time PCR原理與操作定量方法 28
2.5.2 Real-Time PCR之應用 28
第三章 實驗材料與方法 32
3.1 實驗架構 32
3.2 柴油生物降解風化實驗 34
3.2.1 實驗土樣 34
3.2.2 實驗油品 34
3.2.3 實驗操作與採樣方法 34
3.3 土壤中總石油碳氫化合物分析 36
3.3.1 樣品前處理 36
3.3.2 儀器分析 37
3.4 土壤總菌數分析 38
3.5 土壤功能性降解酶分子生物技術分析 39
3.5.1 DNA萃取與聚合酶鏈鎖反應(Polymerase chain reaction, PCR) 39
3.5.2 RNA萃取與反轉錄聚合酶鏈鎖反應(Reverse transcription-PCR, RT-PCR) 41
3.5.3 Real-Time PCR分析 43
3.6 土壤基本性質與肥力分析 47
3.6.1 土壤質地分析 47
3.6.2 土壤酸鹼值測定 50
3.6.3 土壤含水率 50
3.6.4 土壤有機碳含量測定 51
3.6.5 土壤中總磷測定 51
3.6.6 土壤中總氮測定 53
第四章 結果與討論 55
4.1土壤中總石油碳氫化合物濃度變化 55
4.2 土壤中總菌變化 67
4.3 土壤功能性降解酶分析結果 73
4.3.1 土壤功能性降解酶基因表現之定性分析結果 73
4.3.2 土壤功能性降解酶基因表現定量分析結果 84
4.4 氣溫、土壤基本性質與肥力分析結果 114
4.4.1 氣溫 114
4.4.2 土壤質地 115
4.4.3 pH 116
4.4.4 含水量 117
4.4.5 有機碳含量 122
4.4.6 總氮含量 126
4.4.7 總磷含量 130
4.4.8 碳/磷比值與氮/磷比值 133
4.5 比較各因子間相關性 139
第五章 結論與建議 156
5.1 結論 156
5.2建議 158
參考文獻 160


表目錄
表2-1 土壤功能性降解酶作用目標化合物與其降解酶基因 27
表3-1 Real-time PCR實驗中相關降解基因之primer序列 45
表3-2 Real-time PCR實驗中相關降解基因之primer Annealing溫度與預期PCR產物之大小 46
表3-3 Real-time PCR反應條件 46
表4-1中油柴油污染之北部土土壤酶表現總覽 80
表4-2台亞柴油污染之北部土土壤酶表現總覽 81
表4-3中油柴油污染之南部土土壤酶表現總覽 82
表4-4台亞柴油污染之南部土土壤酶表現總覽 83
表4-5受中油柴油污染之北部土(CK)各因子相關性係數 144
表4-6受中油柴油污染之添加有機質北部土(CKO)各因子相關性係數 145
表4-7受中油柴油污染之添加有機質滅菌北部土(CKOS)各因子相關性係數 146
表4-8受台亞柴油污染之北部土(FK)各因子相關性係數 147
表4-9受台亞柴油污染之添加有機質北部土(FKO)各因子相關性係數 148
表4-10受台亞柴油污染之添加有機質滅菌北部土(FKOS)各因子相關性係數 149
表4-11受中油柴油污染之南部土(CY)各因子相關性係數 150
表4-12受中油柴油污染之添加有機質南部土(CYO)各因子相關性係數 151
表4-13受中油柴油污染之添加有機質滅菌南部土(CYOS)各因子相關性係數 152
表4-14受台亞柴油污染之南部土(FY)各因子相關性係數 153
表4-15受台亞柴油污染之添加有機質南部土(FYO)各因子相關性係數 154
表4-16受台亞柴油污染之添加有機質滅菌南部土(FYOS)各因子相關性係數 155


圖目錄
圖2-1烷烴類生物降解途徑(一)與參與之酶的種類 13
圖2-2烷烴類生物降解途徑(二)與參與之酶的種類 14
圖2-3甲苯生物降解途徑(一)與參與之酶的種類 16
圖2-4甲苯生物降解途徑(二)與參與之酶的種類 16
圖2-5多環芳香烴降解途徑 18
圖2-6苯酚、鄰苯二酚類化合物生物降解途徑與參與之酶的種類 21
圖2-7苯酚單加氧酶化學作用示意圖 21
圖2-8鄰苯二酚雙加氧酶化學作用示意圖 21
圖2-9 聯苯生物降解途徑與參與之酶的種類 23
圖2-10聯苯雙加氧酶化學作用示意圖 23
圖2-11萘生物降解途徑與參與之酶的種類 25
圖2-12萘雙加氧酶化學作用示意圖 25
圖3-1實驗架構 33
圖3-2 土壤粒徑三角對照圖 48
圖4-1中油柴油污染北部土樣中總石油碳氫化合物濃度逐月變化 65
圖4-2台亞柴油污染北部土樣中總石油碳氫化合物濃度逐月變化 65
圖4-3中油柴油污染南部土樣中總石油碳氫化合物濃度逐月變化 66
圖4-4台亞柴油污染南部土樣中總石油碳氫化合物濃度逐月變化 66
圖4-5北部土樣之總菌落數逐月變化 72
圖4-6南部土樣之總菌落數逐月變化 72
圖4-7 alkB在各污染土壤中相對於未污染土壤所出現之DNA濃度比值 87
圖4-8 alkB在各污染土壤中相對於未污染土壤所表現之mRNA濃度比值 87
圖4-9 xylE在各污染土壤中相對於未污染土壤所出現之DNA濃度比值 90
圖4-10 xylE在各污染土壤中相對於未污染土壤所表現之mRNA濃度比值 90
圖4-11 nahAc在各污染土壤中相對於未污染土壤所所出現之DNA濃度比值 94
圖4-12 nahAc在各污染土壤中相對於未污染土壤所表現之mRNA濃度比值 94
圖4-13 bphA在各污染土壤中相對於未污染土壤所出現之DNA濃度比值 99
圖4-14 bphA在各污染土壤中相對於未污染土壤所表現之mRNA濃度比值 99
圖4-15 bphA1在各污染土壤中相對於未污染土壤所出現之DNA濃度比值 102
圖4-16 bphC1在各污染土壤中相對於未污染土壤所出現之DNA濃度比值 107
圖4-17 bphC1在各污染土壤中相對於未污染土壤所表現之mRNA濃度比值 107
圖4-18 tmoF在各污染土壤中相對於未污染土壤所出現之DNA濃度比值 111
圖4-19 tmoF在各污染土壤中相對於未污染土壤所表現之mRNA濃度比值 111
圖4-20 實驗期間室內氣溫變化 115
圖4-21實驗期間土壤pH值變化 117
圖4-22實驗期間北部土樣之含水率逐月變化 121
圖4-23實驗期間南部土樣之含水率逐月變化 121
圖4-24實驗期間北部土樣之有機碳含量逐月變化 125
圖4-25實驗期間南部土樣之有機碳含量逐月變化 125
圖4-26實驗期間土壤總氮變化 130
圖4-27實驗期間土壤總磷變化 133
圖4-28實驗期間土壤碳磷比變化 138
圖4-29實驗期間土壤氮磷比變化 138

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