博碩士論文 91224009 詳細資訊




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姓名 宋秉育(Biing-Ywh Song)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 辛基苯酚之分解:分解菌和生物復育之菌相研究
(Degradation of octylphenol: the study of octylphenol-degrading bacteria and bacterial diversity in bioremediation)
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摘要(中) 烷基苯酚聚氧乙基醇為一被廣泛使用的非離子型界面活性劑,其一旦排放至環境中,通常會生成烷基苯酚(Alkylphenol)如壬基苯酚(nonylphenol, NP)、辛基苯酚(octylphenol, OP)等為環境中不易分解,並具有環境荷爾蒙假性雌激素效應的污染有機物,對人體的內分泌與健康具有潛在的威脅。
鑑於目前對於環境中烷基苯酚之生物分解的瞭解仍非常有限,本研究從長期施用農藥的農田土篩選出兩株可生長於辛基苯酚為唯一碳源之細菌,經過BioLog法、脂肪酸圖譜與16S rDNA序列等三種鑑定方法及明膠水解活性測試,其中之一菌株為Pseudomonas nitroreducens OP1,另一菌株為Pseudomonas sp. OP2。此兩菌株皆能以0.001~0.01%的辛基苯酚為唯一生長碳源,其中,以0.005%辛基苯酚培養時,菌株OP1與OP2之比生長率分別為0.17 d-1及0.31 d-1,且培養16天後,菌株OP1與OP2各能去除82%及46%的辛基苯酚,此顯示兩菌株確實能以辛基苯酚為唯一碳源並去除之。此外,兩株分解菌以辛基苯酚為催化基質時,會分別表現5.3及4.4 nmole/min的耗氧活性,以辛基鄰苯二酚為基質時,也有6.1及3.0 nmole/min的耗氧活性,又進一步以西方墨點法分析,兩株細胞粗萃液中皆不具有類似鄰苯二酚2,3-加氧酵素(catechol 2,3-dioxygenase, C23O)的酵素,此顯示此兩菌株在以辛基苯酚為唯一碳源培養時,並不會產生meta-cleavage之開環酵素,但是否存在ortho-cleavage的開環酵素則需進一步研究。
為進一步探討所分離之辛基苯酚分解菌株應用於土壤復育之潛力,本研究另以土壤縮模(microcosm)模擬受污染的土壤環境,除探討以界面活性劑分解菌P. nitroreducens TX1和P. putida TX2分別搭配菌株Pseudomonas sp. OP2添加於含有辛基苯酚聚氧乙基醇的縮模中,對於辛基苯酚聚氧乙基醇及其代謝物辛基苯酚的去除情形,以及添加菌株Pseudomonas sp. OP2於僅含有辛基苯酚的土壤縮模的去除效果外,並利用16S rDNA片段於變性梯度膠體電泳中之指紋,分析上述土壤縮模中微生物菌群之變化及其與污染物降解之關連。其中,菌株P. nitroreducens TX1已證實能分解辛基苯酚聚氧乙基醇,並以辛基苯酚為最終產物,另一菌株P. putida TX2則對於辛基苯酚聚氧乙基醇及辛基苯酚皆具有分解能力。本實驗將各組縮模之土壤混合後,計選殖出24株菌為選殖庫 (Clone library),其中,所含菌群多屬γ-proteobacteria,而選殖菌群內Alcaligenes faecalis (AF155147)、Stenotrophomonas maltophilia (AJ293470)、Aeromonas sp. (AB076858)等三個菌種則曾於文獻中被發表具有烷基苯酚化合物之分解能力。菌群分析結果顯示,三株添加菌株對於縮模內原生菌群均不會造成顯著的影響,其中,原生菌群Herbaspirillum sp. Chnp3-5、Phyllobacterium myrsinace、Brucellaceae bacterium、Stenotrophomonas maltophilia和Aeromonas hydropila為含有辛基苯酚聚氧乙基醇之縮模中的優勢菌,另外,當菌株P. nitroreducens TX1或P. putida TX2分別添加於土壤縮模後,利用液相層析儀分析90天後土壤中辛基苯酚聚氧乙基醇之含量,皆較只含原生菌之縮模提高1.5倍去除能力,亦能利用縮模內辛基苯酚聚氧乙基醇生長成為優勢菌。此外,土壤原生菌中之Aeromonas hydropila、 Pseudomonas sp YG-1、Alcaligenes faecalis為含有辛基苯酚之縮模中的優勢菌,而菌株Pseudomonas sp.OP2添加於縮模後,於四個月操作時間內能一直為優勢菌,且與只含原生菌之縮模比較,約可於90天內提高15-20%之辛基苯酚的移除效果。整體而言,雖然原生菌已有部分的OPEOn降解能力,但藉由添加菌株P. putida TX2及Pseudomonas sp. OP2之組合,可幾乎完全去除外加之辛基苯酚聚氧乙基醇,並對於辛基苯酚也有25%之去除能力,為最具潛力應用於清除辛基苯酚環境污染之菌株。
摘要(英) Alkylphenol polyethoxylates (APEOn) were an extensively-used non-ionic surfactants. When these componds are discharged into natural environment, alkylphenol such as nonylphenol and octylphenol are often found to be as accumulates metabolites. However, alkylphenol is more recalcitrant than APEOn and has been demonstrated as an environmental hormone with estrogenic-like activity. However, only a few studies were reported on the biodegradation of alkylphenol. This study isolated two octylphenol-degrading bacteria from topsoil of farm, which was frequently sprayed by pesticides and surfactant. They were respectively identified as Pseudomonas nitroreducens OP1 and Pseudomonas sp. OP2 by BioLog breathprint, 16S rDNA sequence analysis, fatty acid fingerprint and negative for gelatin hydrolysis activity. Both of them were able to grow on octylphenol (0.001~0.01%) as sole carbon source. The strains OP1 and OP2 showed a specific growth rate of 0.17d-1 and 0.31 d-1 as 0.005% octylphenol as sole carbon source, respectively. After 16 days of cultivation, the strains OP1 and OP2 were able to remove 82% and 46% of octylphenol, respectively. Both strains OP1 and OP2 further revealed an oxygen uptake activity of 5.3 and 4.4 nmole/min respectively using octylphenol as the transformation substrate with 1.5 ml cell suspension of OD=0.3. They also showed 6.1 and 3.0 nmole/min of oxygen uptake rate when octylcatechol as used as a substrate. In addition, Western blotting and C23O dioxygenase activity assay also showed that no catechol 2,3-dioxygenase-like enzyme for the cleavage of aromatic ring was detected in both strains OP1 and OP2 grown on octylphenol as the sole carbon source.
The study further investigated the bioremediation of octylphenol polyethoxylates (OPEOn) and its primary metabolite, OP in soil microcosms with bacteria exogenously added. Two surfactant-degrading bacteria, P. nitroreducens TX1 and P. putida TX2 were respectively added with Pseudomonas sp. OP2 in different OPEOn-contained microcosms to understand the removal of octylphenol polyethoxylates and octylphenol. The strain TX1 was able to degrade OPEOn and formed octylphenol, while the strain TX2 revealed activities to degrade both of OPEOn and OP. The strain Pseudomonas sp. OP2 was further added into an octylphenol-contained microcosm and the variation of octylphenol was then analyzed. The bacterial communities were analyzed by 16S based denaturing gradient gel electrophoresis (DGGE) of rRNA genes by 24 bacterial strains, which were totally cloned from soils in all microcosms. Among these endogenous bacteria, Alcaligenes faecalis (AF155147)、Stenotrophomonas maltophilia (AJ293470)、Aeromonas sp (AB076858) have been found as alkylphenol-degrading bacteria, in precious study. The analysis of bacteria communities indicated that no obvious effect was observed in the endogenous bacteria community when strains OP2, TX1 and TX2 were exogenously added in these microcosms. When P. nitroreducens TX1 and P. putida TX2 were exogenously added in the OPEOn-contained microcosms, both of them were able to enhance the OPEOn removal by 1.5 fold and also found as the dominant bacterial as well as endogenous bacteria such as Herbaspirillum sp. Chnp3-5、Phyllobacterium myrsinace、Brucellaceae bacterium、Stenotrophomonas maltophilia and Aeromonas hydropila. Moreover, the exogenous strain Pseudomonas sp. OP2 was demonstrated to be able to extra remove 15-20% of OP in soil microcosms, and was also the dominant bacteria as well as endogenous strains Aeromonas hydropila、 Pseudomonas sp YG-1 and Alcaligenes faecalis within four months of operation periods. The study showed that endogenous bacteria was able to remove partial OPEOn in microcosms. However, the removal of OPEOn and its metabolite OP were obviously enhanced when strain P. putida TX2 and Pseudomonas sp. OP2 was exogenously added in soil microcosms. Almost all of OPEOn and 25% of OP was able to be removed respectively. These indicated that these two strains, showed the most potential in the application of OP bioremediation.
關鍵字(中) ★ 辛基苯酚
★ 生物復育
★ 變性梯度電泳法
★ 生物分解
★ 生物菌相
關鍵字(英) ★ octylphenol
★ bioremediation
★ DGGE
★ 16S rDNA
★ bacterial diversity
★ biodegradation
論文目次 中文摘要………………………………………………..............................VII
英文摘要………………………………………………..............................V
目錄……………………………………………………………………….VII
表目錄……………………………………………………………………..X
圖目錄……………………………………………………………………..XI
名詞縮寫對照表…………………………………………………………XIII
第一章、前言………………………………………………………………. 1
1.1、烷基苯酚之來源與流布及影響…………………………………….1
1.1.1、非離子性界面活性劑的特性與環境宿命……………………..1
1.1.2、烷基苯酚之環境流布現況……………………………………..2
1.1.3、烷基苯酚代謝物之環境荷爾蒙效應…………………………..3
1.2、烷基苯酚生物分解之研究現況…………………………………….5
1.2.1、烷基苯酚分解菌之篩選………………………………………..5
1.2.2、烷基苯酚化合物之代謝途徑……………………………………7
1.3、分子生物學技術於生物復育之應用……………………………….8
1.3.1、環境污染物生物復育之簡介…………………………………..8
1.3.2、16S rDNA指紋技術於環境微生物研究之應用……………….9
1.3.3、變性梯度膠體電泳法於菌相分析之應用…………………….11
1.3.4、環境中之菌相分析…………………………………………….12
1.4、研究背景與目的……………………………………………………13
第二章、材料與方法……………………………………………………….16
2.1 菌株來源與培養基………………………………………………….16
2.2 菌種鑑定…….………………………………………………………17
2.3 菌種生長特性分析………………………………………………….22
2.4 耗氧酵素活性測定………………………………………………….23
2.5 西方點墨法(Western blotting)……………………………...……….26
2.6 鄰苯二酚生化活性分析………………………………………..….27
.7 高效能液相層析儀分析生物降解產物………………………..…...28
2.8 土壤縮模之菌相分析………………………………..…………….29
2.9親緣演化樹之建立………………....................................................37
2.10 利用高效能液相層析儀檢測土壤中OPEOn與OP之分解情形..38
2.11 化學藥品…………………………………………………..……...39
2.12 儀器設備…………………………………………………..……...39
第三章、結果………………………………………………………………44
3.1辛基苯酚分解菌之菌種鑑定………………………………..……..44
3.1.1 BioLog菌種鑑定結果……………………………………………44
3.1.2 16S rDNA菌種鑑定……………………………………………...45
3.1.3 Fatty acid methyl fringerprint脂肪酸鑑定系統………………….46
3.1.4 明膠水解反應測試 (gelatin hydrolysis)……………...………...46
3.2辛基苯酚分解菌之生長特性………………………………..……..47
3.3 辛基苯酚之代謝特性………………………………………..…….49
3.3.1 菌體耗氧活性分析……………………………..……..………..49
3.3.2 以西方墨點法偵測OP1及OP2之開環酵素…………………52
3.3.3 Catechol 1,2-dioxygenase (C12O)與Catechol 2,3-dioxygenase C23O雙加氧開環酵素活性之偵測............................................53
3.4 辛基苯酚生物復育之菌相分析的產物鑑定....……………..…...54
3.4.1 16S rDNA菌相基因資料庫之建立…..…………………..…….54
3.4.2辛基苯酚分解菌群親源分析…………..…………………..…...55
3.4.3 16S rDNA變性梯度凝膠分析……..………………………..….56
3.4.4原生菌與外添加菌對縮模中之OPEOn與OP分解效果……..57
3.4.5土壤縮模之菌數與菌相分析..……………………………….....58
第四章、討論………………………………………………………………62
4.1菌種篩選及鑑定…………………………………...……………….62
4.2 生長特性與耗氧活性之探討……………………………………….64
4.3代謝途徑之探討……………………………………………………..65
4.4 偵測開環酵素的存在……………………………………………….67
4.5 辛基苯酚生物復育之菌群分析…………………………………….68
第五章、結論………………………………………………………..……..74
第六章、建議………………………………………………………………76
參考文獻……………………………………………………………………77
表……………………………………………………………………………87
圖…………………………………………………………………………..101
附錄…………………………......................................................................131
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丁望賢、吳建誼、周瓊瑤、王正雄 (2000) 壬基苯酚及其相關化合物質在台灣水環境中之分析與調查,第一屆環境荷爾蒙研討會論文集。
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楊嘉蓁 (2001) Triton X-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研究所,碩士論文
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指導教授 黃雪莉(Shir-Ly Huang) 審核日期 2004-12-28
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