博碩士論文 88346006 詳細資訊




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姓名 張怡塘(Yi-Tang Chang)  查詢紙本館藏   畢業系所 環境工程研究所
論文名稱 化學傳輸行為對土壤微生物分解多環芳香烴化合物之影響
(Fates impact on PAH biodegradation in the soil/water system with surfactants)
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摘要(中) 土壤環境中多環芳香烴化合物 (PAH) 之生物復育經常利用界面活性劑淋洗技術。過程中化學傳輸行為與生物分解會同時發生,此時生物分解PAH之作用受界面活性劑之影響而發生促進或抑制現象。本研究選用不含土壤有機質黏土-鈣蒙特石與含有機質(1.883 %)土壤-台中土,及Triton X-100與Brij 35兩種非離子界面活性劑,利用自PAH污染土壤中篩選之微生物與分解PAH之體外酵素laccase分解含不同界面活性劑之水溶液系統與土壤/水系統中之PAH,探討PAH在不同界面活性劑-土壤組合之傳輸行為對生物分解的影響,及分解過程土壤中與溶液中微生物族群之結構與生理生化反應,並探討可能之代謝途徑。
水溶液系統中,PAH之分解速率受到PAH結構之影響,系統中存在之界面活性劑亦可被微生物所利用,成為微生物的碳源。PAH與界面活性劑兩分子間之分子內聚能 (cohesive energy) 可用於解釋PAH與界面活性劑之生物分解。兩分子之內聚能愈接近,代表兩者間之分子鍵結形式相同者愈多,系統中微生物分泌之體外酵素於分解PAH與界面活性劑過程可相互周轉利用,PAH與界面活性劑之分解速率因此會加快。在土壤/水系統中,因受鈣蒙特石顆粒對微生物之干擾作用及PAH與界面活性劑於台中土發生之分佈作用影響,其微生物分解速率大多較水溶液系統慢。當PAH-界面活性劑-土壤/水系統之組合不同時,PAH與界面活性劑之分解速率也不同,並且系統中所含之界面活性劑濃度與結構不同也會造成影響。在微生物族群數量與結構方面,水溶液系統或土壤/水系統之自由態總生菌數都有增加之趨勢,其中假單胞菌屬 (Pseudomonas sp.) 為分解過程之優勢菌,不過所增加之數量受到系統不同PAH種類與土壤性質之影響。Domain Bacteria,phylum/subclass之 α-, β-, γ-Proteobacteria族群於分解過程佔有極高比例,且不受到添加界面活性劑之影響。Brevundimonas (Pseudomonas) diminuta, Caulobacter sp.、Mycoplana bullata, Burkholderia sp.、Pseudomonas aeruginosa等特定族群於水溶液系統或土壤/水系統之自由態佔有高比例,用於負責分解PAH。此外,分解過程之族群生理圖譜 (community-level physiological profiling, CLPP)、代謝潛能與對Biolog之碳源利用率、API ZYM水解酵素反應、代謝途徑等也因組合不同而發生改變。
胞外酵素laccase分解PAH方面,實驗結果顯示,水溶液系統含有Triton X-100與Brij 35之溶液中laccase皆促進PAH的分解作用,又因微胞比單體為更有效之分佈介質,使得含微胞相時的溶液能促使生物分解速率加快。另外由於Brij 35碳氫鏈較Triton X-100碳氫鏈長許多,形成之微胞結構較大,造成較大的立體結構障礙,故含微胞之Triton X-100溶液對PAH分解速率大於Brij 35。鈣蒙特石/水系統中兩種界面活性劑仍具促進分解作用,且酵素可直接利用溶於水溶性微胞與吸附性微胞之PAH,使得當平衡溶液含界面活性劑微胞時之分解速率大於平衡溶液含單體者。分別比較鈣蒙特石/水系統中附著態與自由態分解情形,因PAH與酵素在自由態之質傳速率大於附著態,使得分解行為在自由態中較明顯。進一步將酵素固定在鈣蒙特石上,分解不同界面活性劑溶液的PAH結果顯示,添加Triton X-100有促進分解作用,但添加Brij 35卻有抑制分解之現象,推測原因為鈣蒙特石對Brij 35之吸附量較TX-100少,PAH多進入溶液中微胞,反而阻礙固定化酵素之作用。
摘要(英) Combined bioremediation with surfactant flushing system is believed to be an important process to remove polycyclic aromatic hydrocarbon compounds (PAH) in soil/water systems. Many chemical reactions were generated in the integrated process so as to affect biodegradation. The PAH’s hydrophobicity results in these compounds being strongly sorbed onto soils. The use of surfactants may change the sorption behavior of PAH in soil environment. The aim of this study was to evaluate the fate impacts on PAH biodegradation in soil/water systems with nonionic surfactants. The ability of PAH-biodegraders and fungal laccase in degrading PAHs, naphthalene and phenanthrene, was analyzed. The nonionic surfactants tested were Triton X-100 and Brij 35. Soils selected were composed of a clay (Ca-montmorillonite) and a natural soil (Taichung soil). Bacterial community and physiological profile of free-state and attached microorganisms were determined during biodegradation. A comparative study was also carried out by application of free and immobilized laccases in the soil-water system.
In aqueous, PAHs were mineralized completely but the biodegrading rate was affected by the structure complexity of PAH. PAH-biodegrading bacteria enabled to utilize nonionic surfactant as carbon source in systems. The influence of surfactant additives on PAH biodegradation was successfully evaluated using chemical molecular interaction method, based on the theory of cohesive energy density (CED). Results from this study suggested, PAH have a relatively higher CED value because aromatics compounds with labile π are more polarized to prompt molecular attractions by the induced dipole force. Under different PAH-surfactant compositions, similar CED values are related to facilitate their intermolecular attractions through π-π electron interactions to represent a similar biodegradation pattern. Extracellular enzymatic activity measurements revealed that when induced enzymes targeted same molecular bonding on PAH and surfactant, rapid PAH degradation rate was observed. In soil/water systems,
PAH biodegradation was influenced by the composition of PAH-surfactant-soil/water systems. Particle size of Ca-montmorillonite and the partition of surfactant on Taichung soil played an important role. Rate of biodegradation also was found to be affected by the distribution of PAH in the monomer or micelle surfactant bulk. For bacterial number and diversity, Pseudomonas sp. was dominant during biodegradation although their numbers were affected by PAHs and the soil composition. α-, β-, γ-Proteobacteria of Domain Bacteria had a high percentage. Especially Brevundimonas (Pseudomonas) diminuta, Caulobacter sp., Mycoplana bullata, Burkholderia sp., Pseudomonas aeruginosa took advantage in aqueous or free state in soil/water systems. Moreover, community-level physiological profiling (CLPP), carbon degradation potential, Biolog carbon utilization, API ZYM enzymatic activities even metabolic pathway were alternative in different PAH-surfactant-soil/water systems.
Addition of Triton X-100 and Brij 35 enhanced the biodegradation of aqueous PAH by free laccase. When micelles existed in water systems, PAH biodegradation was greater than that of below critical micelle concentration (CMC). The same results were also found in the Ca-montmorillonite-water system. The phenomena can be ascribed to more amount of PAH partition on into micelles than that of monomers. Micellar phase were to provide microorganisms for extra phase to biodegrade PAH effectively. To compare PAH biodegradation in difference phase, the rate in the aqueous phase was higher than that in the soil phase. On immobilized laccase systems, an inhibited biodegradation in the presence of Brij 35 was observed, and an opposite effect presented in the presence of Triton X-100. Different phenomenon in bioavailability may correlate with smaller Brij 35 sorption on Ca-montmorillonite. PAH was easily into the aqueous Brij 35 micelles, instead of interceptions with immobilized laccase.
關鍵字(中) ★ 分子內聚能
★ 非離子界面活性劑
★ 多環芳香烴化合物 (PAH)
★ 族群生理圖譜 (CLPP)
★ 胞外酵素laccase
關鍵字(英) ★ cohesive energy
★ PAH
★ nonionic surfactant
★ CLPP
★ laccase
論文目次 目次…………………………………………………………………….................頁次
目錄……………………………………………………………………………... I
圖目錄…………………………………………………………………………... VI
表目錄…………………………………………………………………………... X
第一章 前言……………………………………………………………………. 1
1-1 研究緣起………………………………………………………… 1
1-2 研究目的與內容………………………………………………… 3
第二章 文獻回顧……………………………………………………………….. 7
2-1 多環芳香烴化合物……………………………………………… 7
2-1-1 定義與來源……………………………………………. 7
2-1-2 特性……………………………………………………. 7
2-1-3 毒性……………………………………………………. 11
2-1-4 污染現狀………………………………………………. 11
2-2 界面活性劑……………………………………………………… 12
2-2-1 定義…………………………………………………… 12
2-2-2 種類與用途…………………………………………… 12
2-2-3 界面活性劑之性質…………………………………… 12
2-2-4 界面活性劑應用於土壤/地下水有機污染物之復育.. 14
2-2-5 界面活性劑對環境之影響…………………………… 15
2-3 PAH與界面活性劑於土壤/水系統之傳輸行為……………….. 16
2-3-1 傳輸行為描述………………………………………… 16
2-3-2 PAH於土壤/水系統之傳輸行為…………………….. 16
2-3-3 界面活性劑於土壤/水系統之傳輸行為…………….. 17
2-4 生物分解及其生理生化反應…………………………………… 20
2-4-1 PAH之生物分解……………………………………… 20
2-4-2 界面活性劑之生物分解………………………………. 21
2-4-3 界面活性劑對PAH生物分解之影響………………… 23
2-4-4 酵素分解PAH之原理與應用………………………… 25
2-5 微生物族群結構與生理生化特性……………………………… 28
2-5-1 微生物菌群變化………………………………………. 28
2-5-2 族群生理圖譜與生理生化反應………………………. 31
2-6 環境因子對生物分解有機污染物之影響……………………… 34
2-7 有機污染物之傳輸行為與生物分解之關係…………………… 37
2-7-1 PAH之傳輸行為與生物分解之關係…………………. 37
2-7-2 界面活性劑之傳輸行為與生物分解之關係…………. 38
2-8 本章總結………………………………………………………… 39
第三章 研究流程與方法……………………………………………………… .........41
3-1 研究流程………………………………………………………… 41
3-2 研究材料………………………………………………………… 44
3-2-1 多環芳香烴化合物(PAH)…………………………….. 44
3-2-2 界面活性劑……………………………………………. 45
3-2-3 PAH分解菌……………………………………………. 46
3-2-4 土壤……………………………………………………. 48
3-2-5 關鍵酵素………………………………………………. 49
3-3 實驗設計………………………………………………………… 50
3-3-1 PAH分解族群結構與生化特性………………………. 50
3-3-2 添加界面活性劑之水系統中PAH之生物分解……… 52
3-3-3 含界面活性劑之土壤/水系統中PAH之生物分解…... 55
3-3-4 關鍵酵素對PAH分解作用之影響……………………. 59
3-4 研究方法………………………………………………………… 62
3-4-1 PAH、TX-100與DMF之分析方法………………….. 62
3-4-2 Brij35之分析方法……………………………………. 66
3-4-3 菌種鑑定- Biolog®系統……………………………… 66
3-4-4 生菌數分析與菌落培養特徵測定……………………. 68
3-4-5 比攝氧率(SOUR)分析………………………………... 69
3-4-6 細菌生長實驗(OD590)………………………………… 70
3-4-7 水解酵素活性反應分析-APIZYM系統……………… 70
3-4-8 微生物族群結構測定-螢光原位雜交技術(FISH)…… 71
3-4-9 碳源利用情形與代謝潛能分析………………………. 76
3-4-10 族群生理圖譜(CLPP)與群集分析……………………. 77
3-4-11 土壤有機質之生物分解………………………………. 80
3-4-12 自由態(free)與固定化(immobilized)關鍵酵素之
製作.........................................81
3-4-13 關鍵酵素laccase活性分析………………………….. 81
3-4-14 laccase對PAH之分解………………………………… 85
3-4-15 laccase對界面活性劑之分解實驗…………………… 88
第四章 PAH分解菌之族群分佈與生化特性………………………………… .........90
4-1 基質降解能力試驗……………………………………………… 90
4-2 菌種鑑定與族群結構解析……………………………………… 91
4-2-1 菌種鑑定………………………………………………. 91
4-2-2 微生物菌群結構………………………………………. 91
4-3 添加有機溶劑對PAH分解菌之影響…………………………… 96
4-3-1 PAH降解能力比較……………………………………. 97
4-3-2 生物分解含DMF之PAH基質之菌種鑑定…………. 97
4-3-3 生物分解含DMF之PAH基質之菌群分佈…………. 104
4-3-4 系統添加DMF前後之微生物生菌數………………... 104
4-3-5 兩系統微生物於不同PAH碳源生長密度之比較…… 105
4-3-6 族群生理圖譜(CLPP)及其群集關係…………………. 107
4-3-7 碳源利用情形…………………………………………. 111
4-3-8 酵素活性反應…………………………………………. 116
4-4 PAH分解菌代謝反應之推測…………………………………… 121
4-5 本章總結………………………………………………………… 129
第五章 水溶液系統中界面活性劑對微生物分解PAH之影響…………….... 130
5-1 PAH與界面活性劑之生物分解………………………………… 130
5-1-1 PAH之生物分解………………………………………. 130
5-1-2 界面活性劑之生物分解………………………………. 132
5-2 水系統中之界面活性劑對PAH生物分解之影響……………... 134
5-2-1 PAH之生物分解………………………………………. 134
5-2-2 界面活性劑之生物分解………………………………. 135
5-2-3 分子內聚能……………………………………………. 137
5-2-4 應用分子內聚能解釋PAH與界面活性劑之生物分解 140
5-2-5 不同結構界面活性劑對生物分解PAH之影響……… 142
5-3 生物分解過程之微生物族群數量與結構……………………… 144
5-3-1 微生物族群數量………………………………………. 144
5-3-2 微生物族群結構………………………………………. 146
5-4 分解過程之生理生化反應……………………………………… 150
5-4-1 族群生理圖譜(CLPP)與群集分析……………………. 150
5-4-2 碳源利用情形與代謝潛能……………………………. 154
5-4-3 水解酵素活性反應與代謝途徑變化…………………. 158
5-5 含不同界面活性劑laccase對PAH生物分解…………….…….. 165
5-5-1 laccase在含TX-100溶液中對PAH之分解作用……. 165
5-5-2 laccase在含Brij35溶液中對PAH之分解作用……… 167
5-5-3 比較PAH在不同界面活性劑系統之生物有效性…… 169
5-6 本章總結………………………………………………………… 172
第六章 土壤/水系統中界面活性劑對微生物分解PAH之影響…………….. .........174
6-1 土壤/水系統中PAH之生物分解……………………………….. 175
6-1-1 PAH於不同土壤/水系統之生物分解………………… 175
6-1-2 生物分解PAH過程中微生物數量與族群結構之變化 179
6-1-3 生物分解PAH過程之族群生理圖譜(CLPP)與群集分析 183
6-1-4 生物分解PAH過程之生理生化反應………………… 189
6-2 PAH分解菌對界面活性劑之生物分解………………………… 195
6-2-1 界面活性劑於不同土壤/水系統之生物分解………… 195
6-2-2 生物分解界面活性劑過程中微生物數量與族群結構
之變化.......................................198
6-2-3 生物分解界面活性劑過程之族群生理圖譜與群集分
析...........................................203
6-2-4 生物分解界面活性劑過程之生理生化反應…………. 209
6-3 添加界面活性劑對土壤/水系統中PAH生物分解之影響…….. 216
6-3-1 不同土壤/水系統之生物分解速率…………………… 216
6-3-2 生物分解過程微生物數量與族群結構變化…………. 226
6-3-3 生物分解過程之族群生理圖譜(CLPP)與群集分析…. 236
6-3-4 生物分解過程之生理生化反應………………………. 244
6-4 關鍵酵素對鈣蒙特石/水系統中PAH分解作用之影響…….…. 253
6-4-1 鈣蒙特石/水系統中自由態酵素對PAH之分解……... 253
6-4-2 鈣蒙特石/水系統中固定化酵素對PAH之分解……... 258
6-5 本章總結………………………………………………………… 267
第七章 結論與建議……………………………………………………………. 268
7-1 結論……………………………………………………………… 268
7-2 建議……………………………………………………………… 272
參考文獻………………………………………………………………………... 274
附錄A、本研究使用之實驗設備、試劑與培養基
附錄B、本研究之菌落培養特徵
圖目錄
目次………………………………………………………………………………........頁次
圖1-1 本論文研究架構……………………………………………………… 4
圖3-1 本實驗研究流程……………………………………………………… 42
圖3-2 本研究Chemostat示意圖…………………………………………… 47
圖3-3 鈣蒙特石結構示意圖………………………………………………… 48
圖3-4 PAH分解族群結構與生化特性之研究架構流程圖………………… 51
圖3-5 本實驗使用之特殊設計雙耳採樣口血清瓶………………………… 53
圖3-6 本實驗使用於分解單一界面活性劑基質之特殊血清瓶…………… 54
圖3-7 水系統中添加界面活性劑對PAH生物分解之影響研究架構流程圖... 55
圖3-8 添加界面活性劑於土壤/水系統中對生物分解PAH之影響研究架構流程圖 56
圖3-9 關鍵酵素laccase影響PAH分解作用之研究架構流程圖…………. 61
圖3-10 樣品以GC/FID分析PAH之前處理步驟…………………………… .........64
圖3-11 Biolog系統菌種鑑定流程圖………………………………………… 67
圖3-12 比攝氧率(SOUR) 實驗設備示意.圖..................................................... .........69
圖3-13 台中土有機質之生物分解實驗……………………………………… 80
圖3-14 laccase固定化步驟之實驗流程……………………………………… 82
圖3-15 含界面活性劑水系統laccase之相對活性…………………………… 83
圖3-16 土壤/水系統中自由態與固定化酵素活性變化……………………... 84
圖3-17 水系統中自由態酵素對PAH分解之實驗流程……………………... 86
圖3-18 土壤/水系統中自由態酵素對PAH分解之實驗流程………………. 87
圖3-19 Laccase對界面活性劑之分解……………………………………….. 89
圖4-1 單一PAH為基質碳源之微生物生長濃度曲線,微生物基質分別為:
(a)PAH-MSB,(b)PAH-DMF-MSB......................................106
圖4-2 C1與C2 Chemostat系統之(a)族群生理圖譜與(b)群集關係……… 108
圖4-3 Biolog系統分離菌株所得之CLPP,微生物基質分別為:
(a)PAH-MSB;(b)PAH-DMF-MSB…………………………………............109
圖4-4 利用Biolog 分離菌株所得之群集分析,微生物基質分別為:
(a)PAH-MSB;(b)PAH-DMF-MSB......................................110
圖4-5 Biolog GN2培養盤95個碳源之利用情形(培養72 hours)…………. 111
圖4-6 C1與C2 Chemostat系統代謝潛能比較圖………………………….. 112
圖4-7 C1與C2系統API ZYM 酵素活性反應 (培養60小時)…………..... 117
圖4-8 C1與C2系統API ZYM 酵素活性反應之主成份分析……………. .........117
圖4-9 C1系統分離菌株酵素活性反應結果 (單位: nanomole)…………… 119
圖4-10 C2系統分離菌株酵素活性反應結果 (單位: nanomole)…………… 119
圖4-11 主成份分析C1與C2 Chemostat系統分離菌株酵素活性反應……. 120
圖4-12 Chemostat系統之代謝反應推測圖…………………………………... 124
圖4-13 Chemostat系統之醣生與醣解作用代謝反應推測圖………………... 125
圖4-14 Pseudomonas sp. 對應API ZYM之生理生化反應…………………. 127
圖4-15 脂解酶EC No. 3.1.1.3與醣脂質代謝之關係………………………... 128
圖5-1 PAH與界面活性劑在水系統之生物分解…………………………… 131
圖5-2 (a)球狀微胞(b)低水溶性有機物溶於微胞之剖面圖………………... 132
圖5-3 生物分解PAH之降解曲線:(a)NAP(b)PHE(c)PYR及降解速率
μm與比攝氧率比值:(d)NAP(e)PHE(f)PYR............................135
圖5-4 界面活性劑之生物分解……………………………………………… 137
圖5-5 PAH與界面活性劑間之溶解度參數差值…………………………… 141
圖5-6 水系統PAH生物分解之生菌數變化:(a)NAP-Surfactant,
(b)PHE-Surfactant, (c)PYR-Surfactant............................145
圖5-7 PAH生物分解過程之微生物族群結構……………………………… 147
圖5-8 生物分解3種PAH之(a)CLPP與(b)群集分析 (培養72hrs)………. 150
圖5-9 生物分解TX-100之(a)CLPP與(b)群集分析 (培養72hrs)………… 151
圖5-10 生物分解Brij 35之(a)CLPP與(b)群集分析 (培養72hrs)………….. 151
圖5-11 生物分解界面活性劑之(a)CLPP與(b)叢集分析(培養72hrs)………. 151
圖5-12 生物分解3種PAH與2種界面活性劑為碳源之CLPP……………..... 152
圖5-13 生物分解NAP-界面活性劑之(a)CLPP(b)叢集分析………………… 153
圖5-14 生物分解PHE-界面活性劑之(a)CLPP(b)叢集分析………………… 153
圖5-15 生物分解PYR-界面活性劑之(a)CLPP(b)叢集分析………………… 153
圖5-16 生物分解PAH之水解酵素活性反應………………………………... 159
圖5-17 生物分解PAH酵素活性反應之主成份分析………………………... 159
圖5-18 生物分解界面活性劑之水解酵素活性反應………………………… 161
圖5-19 生物分解界面活性劑酵素活性反應之主成份分析………………… 161
圖5-20 生物分解PAH於不同界面活性劑下之水解酵素活性反應……….. 162
圖5-21 生物分解NAP-Surfactant 水解酵素活性反應之主成份分析……… 162
圖5-22 生物分解PHE-Surfactant 水解酵素活性反應之主成份分析……… 163
圖5-23 生物分解PYR-Surfactant 水解酵素活性反應之主成份分析……… 163
圖5-24 水系統含TX-100溶液中laccase 對PAH 之分解(a)NAP(b)PHE…. 166
圖5-25 水系統含Brij 35溶液中laccase 對PAH 之分解(a)NAP(b)PHE….. 168
圖5-26 laccase於水系統對PAH之分解(a)NAP(b)PHE…………………….. 170
圖6-1 土壤/水系統中PAH生物分解……………………………………….. 178
圖6-2 土壤/水系統溶液中分解PAH之生菌數,包括TGE agar
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曾怡禎與張權英 (2001),利用分子生物方法分析微生物社會的結構,環境保護分子生物科技策略論壇 (Ⅲ),行政院環境保護署,科技顧問室。
楊琇婷 (1997),土壤吸附界面活性劑對土壤/水系統中有機污染物傳輸行為之影響,國立中央大學,環境工程研究所,碩士論文。
楊嘉蓁 (2001),TX-100分解菌之分離與分解酵素之特性研究,國立中央大學,生命科學系,碩士論文。
葉佩雯 (2002),分子間作用力影響土壤中非離子有機物傳輸行為之研究,國立中央大學,環境工程研究所,碩士論文。
廖明隆 (1994),界面化學與界面活性劑,再版,文源書局,臺北。
廖萬里 (1996),固定化白腐真菌分解水中多環芳香族碳氫化合物之研究,國立中央大學,環境工程研究所,博士論文。
趙承琛 (2000),界面基礎科學,第十九版,復文書局。
劉芹君 (2000),淡水沙崙海岸耐寒細菌的分離與鑑定,國立陽明大學,微生物暨免疫學研究所,碩士論文。
蔡幸真與陳啟祥 (1994),Biolog 菌種自動化鑑定系統對環境中革蘭氏陰性菌之使用評估,中國環境工程學刊,第4卷,第3期。
蘇慧慈 (1996),原位分子生物學技術,徐氏基金會,台北。
指導教授 李俊福(Jiunn-Fwu Lee) 審核日期 2006-5-1
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