臺灣博碩士論文加值系統

本研究主要探討氨基甲酸鹽類農藥納乃得(Methomyl)、加保扶(Carbofuran)及加保利(Carbaryl)與有機磷類農藥托福松(Terbufos)對河川生物膠膜中細菌與藻類族群的分佈特性的影響，並且利用生物歧異度指標及分子生物技術了解其受農藥影響之多樣性變化與鑑定優勢族群，以獲得對該農藥具耐受性或敏感性之微生物種類。
春季對於農藥耐受能力較好的矽藻藻種為，Cyclotella radiosa (CRAD) 及 Gomphonema parvulum (GPAR) 佔所有矽藻比例約74%以上，而Navicula vermicularis (NVER) 與Sellaphora bacillum (SEBA)，屬於對農藥敏感的藻種，春季矽藻對於加保利的耐受能力較低，在添加含有此一農藥時歧異度就會降低；夏季主要優勢藻為 Nitzschia angustata (NIAN) 及 Navicula veneta (NVEN)，對於農藥耐受能力較好的矽藻為Cymbella silesiaca (CSLE)，其在農藥添加之後此藻種數量增加，有成為優勢藻種的趨勢，Nitzschia linearis (NZLT) 屬於對農藥較敏感之藻種，夏季的矽藻族群歧異度隨著實驗天數增加而有升高的情形，可見其對於農藥耐受性較高，；秋季對於農藥耐受能力較高的藻種為Cyclotella distinguenda (CDTG) 、Cymbella silesiaca (CSLE) 與 Navicula veneta (NVEN)，而Navicula ventricosa (NNEO) 對於農藥加保扶的耐受能力較低，在添加此一農藥時數量均有減少的情形，秋季的矽藻則是對於混合農藥較敏感，加入混合農藥之後歧異度降低；冬季優勢藻種為主要為Nitzschia dissipata (NDIS) 與Navicula trivialis (NTRV) ，在農藥添加之後數量減少的藻種為Gomphonema parvulum (GPAR)與 Nitzschia capitellata (NCPL)，冬季矽藻族群在農藥添加之後歧異度有下降。
從變性梯度凝膠電泳(DGGE)圖譜的條帶數目與圖譜統計分析，再經由基因序列比對與分析結果，發現春季細菌種類較少，因添加農藥加保扶之關係，在實驗第10天時，出現Deltaproteobacteria此類菌種能抵抗不良環境，因此能對於農藥具有較好的耐受能力，使樣本間相似度變高；夏季樣本在農藥加入時條帶數目變少，對於農藥具有耐受能力的細菌存活下來，而使樣本間的相似度增加，其中對於農藥耐受能力較好的菌種為Microbacterium sp. EMB-1；秋季樣本對於混合農藥納乃得與加保扶較敏感，當農藥添加之後不僅細菌種類變少，含有此兩種農藥的樣本相似度相對的提高，其中對於農藥耐受能力較好的菌屬為Alphaproteobacteria；冬季樣本中在添加農藥之後各樣本間的相似度都變低，細菌種類則有增加的趨勢，在添加農藥之後即出現並成為優勢菌的是Firmicutes中的Clostridia。
總之，河川生物膠膜內之矽藻與細菌族群結構隨著季節不同而不同，其對農藥之耐受程度亦不同，且耐受矽藻與細菌種類亦明顯不同，可見不同季節生長之河川生物膠膜對河川自淨能力有不同之貢獻，未來可進一步篩選這些具農藥耐受力之微生物種類來分析其農藥降解能力，以應用於農藥污染水體之生物復育。

This study investigated the effects of carbamate pesticides Methomyl, Carbofuran and Carbaryl and organophosphate pesticide Terbufos to bacterial and algal community structures in river biofilms. Diversity index and molecular biological techniques were used to determine the changes in microbial diversity and to identify dominant species in order to obtain the tolerant and sensitive microbial species.
The diatom species like Cyclotella radiosa (CRAD) and Gomphonema parvulum (GPAR) were not affected by pesticide exposure with the ratio of approximately 74% of total diatom biomass, while Navicula vermicularis (NVER) and Sellaphora bacillum (SEBA) belonged to the pesticide-sensitive species in spring. The diatom diversity index in spring decreased after exposure to carbaryl, indicating diatom in spring river biofilms were less tolerant to this pesticide. In summer, the dominant diatoms were Nitzschia angustata (NIAN) and Navicula veneta (NVEN). Cymbella silesiaca (CSLE) particularly increased and nearly became dominant species after adding pesticides, demonstrating it was a pesticide-tolerant species in summer. Nitzschia linearis (NZLT) was sensitive to the pesticides in this season. The diatom diversity index in summer increased after adding pesticides, suggesting the diatom community within biofilms had high tolerance to pesticides. In autumn, Cyclotella distinguenda (CDTG), Cymbella silesiaca (CSLE) and Navicula veneta (NVEN) were pesticide tolerant species. After adding carbofuran, Navicula ventricosa (NNEO) decreased, demonstrating it was carbofuran-sensitive species. The diatom diversity index decrease after adding the mixtures of two or three pesticides, indicating diatoms from autumn were sensitive to these types of pesticides. The dominant species in winter were Nitzschia dissipata (NDIS) and Navicula trivialis (NTRV), while the pesticide-sensitive species were Gomphonema parvulum (GPAR) and Nitzschia capitellata (NCPL). The diatom diversity index in winter decreased after adding pesticides. The bacterial diversity and identification were analyzed by denaturing gradient gel electrophoresis (DGGE), cloning and sequencing. The results showed the number of bacterial species was low in spring. After adding carbofuran for ten days, Deltaproteobacteria appeared in experimental groups containing carbofuran, resulting in high similarity between these groups. This species could grow in harsh environment and may also show better tolerance to this pesticide. In summer, samples containing pesticides had less bands in DGGE gels than those without pesticides. The tolerant bacterial species survived after adding pesticide, resulting in high similarity between samples containing pesticides. The pesticide-tolerant species was Microbacterium sp. EMB-1. Baterial samples from autumn were more sensitive to the mixture of methomyl and carbofuran, resulting in decreasing in bacterial species and increasing similarity between samples. Alphaproteobacteria were pesticide-tolerant species in autumn. After adding pesticides, similarity of bacterial communities between samples was low, but the number of bacterial species increased. The dominant bacterial species were Firmicutes Clostridia after adding pesticide.
To sum up, the diatom and bacterial community structures within river biofilms differed with season and in their tolerance to pesticides. There were significant differences in pesticide-tolerant diatom and bacterial species between seasons, demonstrating river biofilms from different seasons had different contribution to river self-purification. Screening these pesticide-tolerant microbial species from river biofilms should be further conducted to analyze their degradability of pesticides and to apply in bioremediation of pesticide-contaminated water.

摘要 I
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河川生物膠膜的介紹 5
2.2河川生物膠膜的組成與重要性 7
2.2.1矽藻的介紹與應用 9
2.2.2細菌的介紹與應用 12
2.3環境微生物菌群結構的分析技術介紹 15
2.3.1 16S rDNA在細菌演化分類與鑑定上的獨特性及應用 19
2.3.2 PCR-DGGE技術之介紹與應用 20
2.3.2.1 DNA萃取技術 21
2.3.2.2 聚合酶鏈鎖反應 22
2.3.2.3 DGGE技術介紹 23
2.4氨基甲酸鹽類與有機磷類農藥簡介 24
2.4.1 納乃得 25
2.4.2 加保扶 26
2.4.3 加保利 27
2.4.4 托福松 28
3.1 藥品、儀器與培養基 30
3.1.1 使用之藥品與其來源 30
3.1.2 實驗儀器 32
3.2河川生物膠膜附著架之製作與採樣 34
3.3實驗設置與樣本採取 34
3.4農藥添加後河川水質分析 38
3.5矽藻玻片製作與計數 40
3.5.1建立矽藻指標 41
3.5.2 歧異度計算 41
3.6河川生物膠膜內細菌族群結構分析 42
3.6.1河川生物膠膜內細菌之培養與計數 42
3.6.1.1微生物培養基配置 42
3.6.1.2細菌培養與計數方法 42
3.6.2河川生物膠膜內細菌基因組DNA萃取 43
3.6.3河川生物膠膜內細菌基因組DNA檢視 44
3.6.4河川生物膠膜內細菌16S rDNA增幅─巢式聚合酶連鎖反應(nested-PCR) 45
3.6.5河川生物膠膜內細菌16S rDNA片段電泳檢視 47
3.6.6河川生物膠膜內細菌16S rDNA片段純化與濃縮 48
3.6.7變性梯度膠體電泳(DGGE) 49
3.6.7.1變性梯度凝膠電泳膠體製作與操作步驟 49
3.6.7.2 DGGE Fingerprinting pattern 分析 52
3.6.8 PCR-16S rDNA片段之基因選殖(cloning) 53
3.6.9序列資料處理 56
第四章 結果與討論 57
4.1水質分析結果 57
4.2不同季節之河川生物膠膜內矽藻族群結構於農藥添加前後之變化 77
4.3不同季節之河川生物膠膜內細菌總數於農藥添加前後之變化 95
4.4不同季節之生物膠膜內細菌族群結構於添加農藥前後之變化 106
4.4.1 河川生物膠膜內微生物16S rDNA片段nested-PCR擴增 106
4.4.2氨基甲酸鹽類農藥對河川生物膠膜內微生物多樣性之探討 107
4.4.2.1 河川生物膠膜內微生物DGGE電泳圖與比對結果 107
4.4.2.2 DGGE電泳圖譜統計分析結果 118
4.4.2.3 DGGE核酸條帶基因轉殖結果 129
4.4.3 序列分析 134
4.4.3.1河川生物膠膜內微生物基因序列比對與分析結果 134
4.4.3.2 河川生物膠膜內微生物基因序列親源關係 140
第五章 結論與建議 144
5.1結論 144
5.2建議 148
參考文獻 149

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