臺灣博碩士論文加值系統

為避免水質的優養化，以工程方法來減少廢水中磷的濃度便成為廢水處理的重要課題之一。若考慮經濟性、可靠、及高效率的特性，生物除磷被建議為最適合的除磷方式。在目前廣為使用的程序為EBPR(Enhanced Biological Phosphorus Removal)生物除磷程序中，雖然有幾類分離出的微生物菌株被認定為聚磷酸鹽累積菌，但實際參與此反應的微生物種類以及以生長特性至今尚未有所定論。而文獻中曾指出，某些以醋酸為基質之除磷反應槽的菌群中有80％為演化上接近Rhodocyclus (Rhodocyclus-related)的菌種，且由最近學者發表之研究中得知此類微生物在實廠活性污泥中所佔比例可達10％以上，但此類Rhodocyclus-related的菌種是否為紫色不含硫光合作用細菌之一，或是紫色不含硫光合作用細菌與Rhodocyclus-related的菌種在EBPR生物除磷程序中所扮演的角色及累積聚磷酸鹽的能力也尚未明瞭。
本研究欲瞭解紫色不含硫光合作用細菌或演化分類上接近紫色不含硫光合作用細菌之菌株在活性污泥生物除磷系統中所可能扮演的角色。實驗設計上，先以傳統培養純化並作菌株計數比較一般處理程序與EBPR處理程序中紫色不含硫光合作用細菌之菌數及菌相，再配合分子生物方法，包含螢光原位雜交(FISH)、聚合脢連鎖反應(PCR)、瓊脂糖電泳(agarose gel electrophoresis)、變性梯度凝膠電泳(DGGE)…等方法進行不同來源污泥槽中紫色不含硫光合作用細菌之比較分析。
初步結果發現，紫色不含硫光合作用細菌在活性污泥槽中之總菌數較文獻中提及之100,000 cells/mL少兩個數量級。光譜分析發現，分離出之純菌約90％以上較趨向於Rhodopseudomonas屬；經電泳分析結果發現，一般僅具曝氣程序之活性污泥中紫色不含硫光合作用細菌之種類較複雜，但總數較少，約1,000 cells/mL以下；而EBPR除磷程序中紫色不含硫光合作用細菌之種類較為單純，但總菌數較多，約3,000 cells/mL至6,000 cell/mL之間。
由PCR-DGGE的結果發現，一般僅具曝氣程序的反應槽活性污泥菌相，比厭氧好氧交替程序(如EBPR程序)的反應槽活性污泥菌相複雜。活性污泥中所純化出之紫色不含硫光合作用細菌菌種以Rhodopseudomonas palustris最常見，此外尚有Rhodoplanes elegans 、Rhodobacter blasticus及 Rhodobacter capsulatus，此外亦分離出紫色含硫光合作用細菌Allochromatium vinosum。而在生物除磷程序的反應槽活性污泥中，最易分離出的為Rhodopseudomonas palustris。但在純化分離純菌試驗中，並未成功分離出Rhodocyclus，故尚無法探究此類菌種累積聚磷酸鹽的能力。

Wastewater phosphorus removal using engineering strategies is one of the important subjects on eutrophication control. In all the available methods, biological phosphorus removal becomes very popular because its low cost, good reliability, and high efficiency. Even though several groups of microorganisms had been isolated from the enhanced biological phosphorus removal (EBPR) process and their ability on accumulating polyphosphate had been confirmed, there is no conclusion on what microorganism plays the major role on the biological phosphorus removal process. From the literatures, more than 80% bacteria in some acetate-feeding phosphorus removal reactors were found to be closely related to Rhodocyclus (belonging to purple non-sulfur phototropic bacteria). Furthermore, the number of these Rhodocyclus - related microorganisms were found to be above 10% in some full-scale wastewater treatment plant. However, the role of purple non-sulfur phototropic bacteria or these Rhodocyclus — related bacteria in the full-scale EBPR process as well as their ability on polyphosphate accumulating is still not clear.
The objectives of this study are to understand the possible roles of purple non-sulfur phototropic bacteria or these Rhodocyclus — related bacteria in the full-scale EBPR processes. Traditional isolation and numeration methods for purple non-sulfur bacteria were applied first to compare the difference between sludge samples from normal activated sludge process and EBPR process. After that, molecular methods including fluorescence in situ hybridization (FISH)、polymerase chain reaction (PCR)、agrose gel electrophoresis、denauring gradient gel electrophoresis (DGGE) were used for further bacterial community comparisons.
It was shown that the number of purple non-sulfur bacteria is 100 times lower than the previously reported 100,000 cfu/mL. Absorption spectrums of isolated pure cultures showed that more than 90% of purple non-sulfur bacteria isolated were similar to Rhodopseudomonas. Furthermore, numeration result combined with DGGE analysis demonstrated that sludge sample from EBPR process had more purple non-sulfur bacteria (3,000 cells/ml — 6,000 cells/ml) than from normal activated sludge (lower then 1,000 cells/ml). Furthermore, even though normal activated sludge sample had fewer purple non-sulfur bacteria, the composition of these bacteria were more complicated than sample from EBPR process.
Isolated purple non-sulfur bacteria were identified by gene extraction and sequencing. Four major species were found in the isolated pure cultures : Rhodopseudomonas palustris、Rhodoplanes elegans、Rhodobacter blasticus and Rhodobacter capsulatus. Among them, Rhodopseudomonas palustris is the most frequently isolated one. One purple sulfur bacteria identified as Allochromatium vinosum was also isolated by the same method used for culturing purple non-sulfur bacteria. However, no pure culture of Rhodocyclus or organisms closely relate to Rhodocyclus were isolated.

目 錄
中文摘要………………………………………………………………………Ⅰ
中文摘要………………………………………………………………………Ⅲ
目錄…………………………………………………………………………………Ⅴ
表目錄………………………………………………………………………………Ⅸ
圖目錄…………………………………………………………………………XI
第一章 前言…………………………………………………………………………1
1-1 研究緣起……………………………………………………………………1
1-2 研究目的…………………………………………………………………3
第二章 文獻回顧…………………………………………………………………5
2-1 活性污泥法……………………………………………………………… …..5
2-1-1 標準活性污泥法……………………………………………………..5
2-1-2 活性污泥中之菌相組成……………………………………………..5
2-2 生物除磷……………………………………………………………………..9
2-2-1 生物除磷之機制及原理……………………………………………..9
2-2-2 EBPR系統中微生物菌相………………………………………....10
2-3 光合作用細菌……………………………………………………………….12
2-3-1 光合作用細菌獲得能量之方式……………………………………12
2-3-2 無氧光合營微生物…………………………………………………17
2-3-3 光合營微生物之種類及其特性……………………………..……..18
2-3-4 紫色不含硫光合作用細菌…………………..……………………..22
2-3-4-1 紫色不含硫光合作用細菌生理特性…….…………………..22
2-3-4-2 紫色不含硫光合作用細菌鑑定方法………..……………….22
2-3-4-3 紫色不含硫光合作用細菌之應用………………..………….25
2-3-5 光合作用細菌在環工上之相關研究與應用………………………26
2-4 傳統方法分析菌相…………………….……………………………………28
2-4-1 傳統鑑定菌種之方法……………………………...……………….28
2-4-2 傳統方法所產生之偏差………………………………...………….29
2-4-2-1 EBPR系統之偏差…………………………………………..29
2-4-2-2 硝化系統之偏差…………………………………………...…30
2-5 分子生物技術於鑑定分析菌相之突破…………………………………….32
2-6 分子生物技術於環境工程上之應用……………………………………….34
2-6-1 16S rDNA………………………………………………………….35
2-6-3 螢光原位雜交法……………………………………………………36
2-6-3 聚合酶連鎖反應….………………………….……………………..39
2-6-4 電泳分析……………………………….…….……………………..40
2-6-4-1 瓊脂糖凝膠電泳……………………….………….………….40
2-6-4-2 變性梯度凝膠電泳………………………………..………….40
第三章 材料與方法…………………………………………………………………43
3-1 實驗架構…..………………………………………..……………………….43
3-2 實驗設備…………………………………………………………………….44
3-3 活性污泥來源……………………………………………………………….45
3-4 純菌培養、分離與測試……………………………………………………..46
3-4-1 培養基……………………………………………………………....46
3-4-2 各污水廠活性污泥中紫色不含硫光合作用細菌之計數…………48
3-4-3 純菌篩選與分離純化………………………………………………48
3-4-4 純菌株吸收光譜分析………………………………………………49
3-5 環境樣本菌群分析…………………………………………………………50
3-5-1 螢光原位雜交法……………………………………………………50
3-5-1-1 固定………………………………………………………..….50
3-5-1-2 雜交………………………………………………….………..51
3-5-1-3 清洗……………………………………...……………………51
3-5-1-4 顯像………………………………………….….…………….51
3-5-2 DAPI染色……………………………………………….……..….52
3-6 各水廠樣本與純菌之分析比較…………………………………………….54
3-6-1 DNA萃取………………………………………………………….54
3-6-1-1 純菌DNA之萃取……………………………………...……..54
3-6-1-2 環境樣本DNA之萃取……………………...………………..55
3-6-2 聚合酶連鎖反應……………………………………………………56
3-6-2-1 pufM引子……………………………………………………54
3-6-2-2 16S rDNA primer sets ……………………………...………56
3-6-2-3聚合酶連鎖反應使用藥品………………………….………..57
3-6-2-4 操作條件…………………………………………………...…57
3-6-3 電泳分析…………………………………………..………………..58
3-6-3-1 瓊脂膠糖凝膠電泳…………………..……………………….58
3-6-3-2 變性梯度凝膠電泳……………………………………...……59
第四章 結果與討論…………………………………………………………………60
4-1 傳統培養分析方法…………………………………………………………61
4-1-1 各水廠中紫色不含硫光合作用細菌數量…………………………61
4-1-2 純菌分離純化……………………………...……………………….64
4-1-3 純菌光譜分析結果………………………………...……………….65
4-2 螢光原化雜交法分析活性污泥菌相結構………………….………………67
4-3 聚合酶連鎖反應及電泳法分析…………………………………………….81
4-3-1 聚合酶連鎖反應、電泳分析及定序……………………………….81
4-3-2 引子pufM primers測試……………………………….……………82
4-3-2-1 環境樣本測試………………………………………..……….82
4-3-2-2 純菌測試………………………………………………..…….83
4-3-3 變性梯度膠測試……………………………………………...…….84
4-3-3-1 混合純菌測試…………………………………………..…….84
4-3-3-2 環境樣本再現性測試………………………...………………85
4-3-4 以pufM primers分析各水廠間活性污泥………………….……....86
4-3-5 以pufM primers分析比較活性污泥與分離純菌…………….……91
4-3-5-1 南投中興新村中正污水處理廠…………………………….91
4-2-5-2 暨南大學SBR處理廠………………………………………95
4-3-5-3 暨南大學A2O處理模廠厭氧槽……………………………..99
4-3-5-4 暨南大學A2O處理模廠無氧槽…………………………….102
4-3-5-5暨南大學A2O處理模廠好氧槽…………….…………….105
4-3-5-6 分析結果誤差探討………………………………………….108
4-4 純化及定序結果…………………………………………………...………110
4-4-1 活性污泥樣本……………………………………………………110
4-4-2 純菌…………………………………………………………..…..111
4-4-2-1純菌株pufM gene定序結果………………………….112
4-4-2-1-1 中正污水處理廠活性污泥分離之純菌………………113
4-4-2-1-2 暨南大學污水處理廠活性污泥分離之純菌…………116
4-4-2-1-3 暨南大學A2O模廠厭氧槽活性污泥分離之純菌…118
4-4-2-1-4 暨南大學A2O模廠無氧槽活性污泥分離之純菌…120
4-4-2-1-5 暨南大學A2O模廠好氧槽活性污泥分離之純菌…122
4-5 關於Rhodocyclus…………………………………………………………126
第五章 結論與建議………………………………………………………………129
5-1 結論…………………………………………………………………...……129
5-2 建議…………………………………………………………..…………….131
參考文獻……………………………………………………………………………133
表 目 錄
表2-1 活性污泥中常見之微生物相………………………………………………....8
表2-2 葉綠素a與Bchl a,b,c,d,e中R1-R7之成分…….…………………………..16
表2-3 光合作用細菌在自然界中之分佈…………………………………………..17
表2-4 光合營生物之代謝形式……………………………………………………..20
表2-5 紫色不含硫光合作用細菌對不同碳源利用情形…………………………..23
表2-6 文獻中常使用的寡核苷酸探針……………………………………………..38
表3-1 活性污泥來源及其處理程序………………………………………………..45
表3-2 Rhodospririllaceae培養基成分…………………………………………...46
表3-3 Trace element solution SL7成分…………………………………………...47
表3-4 吸收光譜操作條件…………………………………………………………..49
表3-5 不同菌綠素之最大吸光度…………………………………………………..49
表3-6 各濾鏡組所適用之螢光染劑與基本資料…………………………………..53
表3-7 本實驗中所使用之寡核苷酸探針…………………………………………..53
表3-8 各緩衝液成分………………………………………………………………..53
表3-9 Formamide濃度與NaCl濃度對照表…………………………………….54
表3-10 pufM primers之目標族群及其序列……………………………………...56
表3-11 16S rDNA primers之目標族群及其序列…………………………….…56
表3-12 DNA序列長度對應所需之瓊脂糖膠體濃度…………………………….58
表3-13 變性梯度膠所使用之各化學物質…………………………………………59
表4-1 各水廠中紫色不含硫光合作用細菌之計數結果…………………………..61
表4-2 各處理廠活性污泥中純化紫色不含硫光合作用細菌數目………………..64
表4-3 純菌光譜分析結果…………………………………………………………..65
表4-4 各污水處理廠活性污泥菌相結構…………………………………………..68
表4-5 菌數及菌相比較……………………………………………………………..71
表4-6 DGGE分析活性污泥樣本獲得之亮帶...…………………………………..88
表4-7 紫色不含硫光和作用細菌數量與菌種關係表……………………………..89
表4-8 各污水處理廠活性污泥分離純化菌株之定序編號………………………111
表 4-9 pufM primer sets設計來源……………………………………………..112
表 4-10 中正污水處理廠活性污泥分離之各純菌定序及比對結……………..114
表4-11 暨南大學污水處理廠活性污泥分離之各純菌定序及比對結果……….117
表 4-12 暨南大學A2O模廠厭氧槽活性污泥分離之各純菌定序及比對結果….119
表 4-13 暨南大學A2O模廠無氧槽活性污泥分離之各純菌定序及比對結果.121
表 4-14 暨南大學A2O模廠好氧槽活性污泥分離之各純菌定序及比對結果…123
表 4-15各污水處理廠純化出之菌種比對結果……………………………….....124
圖 目 錄
圖2-1 標準活性污泥處理程序示意圖……………………………………………....6
圖2-2 葉綠素a與菌綠素a結構上之差異………………………………………...12
圖2-3 葉綠素a與Bchl a,b,c,d,e構造圖………………………………………….15
圖2-4 光合菌之分類示意圖………………………………………………………..21
圖2-5 變性梯度凝膠電泳原理……………………………………………………..41
圖2-6 變性梯度凝膠電泳呈像圖……………..……………………………………42
圖3-1 文獻中三菌種之光譜圖……………………………………………………..49
圖3-2 聚合酶連鎖反應操作條件示意圖……………………………………..……57
圖4-1 各廢污水處理廠活性污泥菌相組成柱狀圖…………………………...…69
圖4-2 台中酒廠污水處理廠活性污泥FISH結果……………………………….73
圖4-3 台中工業區聯合污水處理廠活性污泥FISH結果……………………….74
圖4-4 福田水資源回收中心活性污泥FISH結果……………………………….75
圖4-5 中興新村中正污水處理廠活性污泥FISH結果………………………….76
圖4-6 暨南大學SBR處理實廠活性污泥FISH結果……………….…………77
圖4-7 暨南大學A2O模廠厭氧槽活性污泥FISH結果………………………..78
圖4-8 暨南大學A2O模廠無氧槽活性污泥FISH結果………………………..79
圖4-9 暨南大學A2O模廠好氧槽活性污泥FISH結果………………………..80
圖4-10 環境樣本測試pufM primers……………………………………………...82
圖4-11 分離純菌株測試pufM primers……………………………………………83
圖4-12 混合純菌之電泳分析……………..………………………………………..84
圖4-13 環境樣本進行DGGE之再現性……………………….………………….85
圖4-14 以瓊脂糖凝膠電泳分析各污水廠活性污泥…………...………………….86
圖4-15以變性梯度凝膠電泳分析各污水廠活性污泥………..…………………..87
圖4-16亮帶相對位置概略圖(圖4-15)....……….………………….……………..87
圖4-17以瓊脂糖凝膠電泳分析中正污水處理廠分離出之18株純菌…....…….91
圖4-18 以變性梯度凝膠電泳分析比對中正污水處理廠活性污泥與其純化出之13株純菌……………………………………………………………………...92
圖4-19亮帶相對位置概略圖(圖4-18)…..………………………….……………..92
圖4-20以瓊脂糖凝膠電泳分析暨南大學SBR處理廠所分離出之15株純菌…95
圖4-21變性梯度凝膠電泳分析比對暨南大學SBR活性污泥及其9株純菌…96
圖4-22亮帶相對位置概略圖(圖4-21)…..………………………….……………..96
圖4-23瓊脂糖凝膠電泳分析暨南大學A2O模廠分離厭氧階段之18株純菌…99
圖4-24變性梯度凝膠電泳比對暨南大學A2O模廠厭氧槽中活性污泥及11株純菌……………………………..……………………………………...100
圖4-25亮帶相對位置概略圖(圖4-24)………...…………………..……………..100
圖4-26瓊脂糖凝膠電泳分析暨南大學A2O模廠分離無氧階段之13株純菌…102
圖4-27變性梯度凝膠電泳比對暨南大學A2O模廠無氧槽中活性污泥及7株純菌………………………………………………………………………...103
圖4-28亮帶相對位置概略圖(圖4-27)...…….…………………….……………..103
圖4-29瓊脂糖凝膠電泳分析暨南大學A2O模廠分離好氧階段之7株純……105
圖4-30變性梯度凝膠電泳比對暨南大學A2O模廠好氧槽中活性污泥及7株純菌………………………………………………………………………...106
圖4-31亮帶相對位置概略圖(圖4-30)…..……..………………….……………..106

國內文獻
張怡塘、林瑩峰、章裕民、方鴻源、邱應志、袁又罡 (1997)，環境微生物，中華民國環境工程學會。
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