臺灣博碩士論文加值系統

金（Au）和鈀（Pd）等貴金屬廣泛的應用在工業和生物醫學上。由於這些貴金屬日益稀少，因此從二次資源（廢棄物）回收這些貴金屬便成為令人感興趣且重要的研究領域。利用微生物可吸附金屬的特性，或許能開發使用微生物從工業廢棄物回收這些貴金屬。然而，這些金屬離子通常亦具有抗微生物特性，因此探討這些貴金屬的生物吸附的應用需先篩選及培養對這些金屬具有耐受高濃度的微生物。
首先，本實驗從重金屬汙染的土壤中進行篩菌，分離出兩株可耐受高濃度鈀的本土原生微生物菌株，經過16s rRNA基因進行定序比對後將其命名為Ochrobactrum sp. strain 71與Microbacterium sp. strain 73。 接著，比較這兩株菌與本研究室先前所分離出的重金屬抗性菌Pseudomonas sp. EJ01對貴金屬鈀的耐受性進行比較，結果顯示此三株菌皆可生長於含有180-270 ppm鈀離子的Luria-Bertani（LB）培養基中，其中Ochrobactrum sp.與 Microbacterium sp. 更可耐受至360 ppm的鈀離子。經由鈀離子感應耦合電漿原子發射光譜法（NIEA W311.53C）檢測發現，當微生物生長在含鈀的環境中，約可吸附總鈀濃度的25%。根據文獻指出微生物對於貴金屬的吸附能力大都來自於胞外多醣體之功能，故本研究直接利用從Ochrobactrum sp.與Microbacterium sp.所萃取出的胞外多醣體單獨進行鈀的吸附實驗，發現胞外多醣體確實對鈀具有明顯的吸附效果，約10-15%。最後，再將這兩株菌的胞外多醣體進行醣類組成分析。

Precious metals such as gold (Au) and palladium (Pd) are widely used in industrial and biomedical applications. Recovery of these precious metals has become an area of particular interest. Biosorption of these rare metals is of the promising approach for recovery of these metals from industrial wastes. However, these metal ions have also been recognized to possess antimicrobial properties. Microorganisms able to tolerate high concentrations of these metals may facilitate the application of biosorption of these precious metals We evaluate several heavy metal resistant strains including Pseudomonas aeruginosa EJ01 previously isolated from our laboratory for their ability to tolerate Pd when grown in general complex medium such as Luria-Bertani (LB) medium. These strains could grow in LB medium containing Pd ranging from 180 to 270 ppm. In addition to these heavy metal resistant strains, we also isolated two palladium resistant microorganisms from soil contaminated with high concentrations of heavy metals. The two isolates were able to tolerate Pd up to 360 ppm. Analysis of 16S rRNA gene sequences led to the identification of these two isolates as Ochrobactrum sp. and Microbacterium sp. and were designed as Ochrobactrum sp. strain 71 and Microbacterium sp. strain 73, respectively.
It was found that about 25% of the total palladium concentration could be adsorbed when the microorganisms grown in a palladium-containing medium by ion inductively coupled plasma atomic emission spectrometry (NIEA W311.53C). According to the literature, the adsorption capacity of microorganisms for noble metals is mostly derived from the function of extracellular polysaccharides. Therefore, we also examined the extracellular polysaccharides extracted from Ochrobactrum sp. and Microbacterium sp. for palladium adsorption. The polysaccharide showed a significant adsorption effect on palladium, about 10-15%. Finally, the extracellular polysaccharides of the two strains were subjected to saccharide composition.

書名頁 I
摘要 II
ABSTRACT IV
誌謝 VI
目錄 VII
表目錄 XII
圖目錄 XIII
第一章、 前言 1
1.1 簡介 1
1.2 研究動機 2
第二章、 文獻回顧 3
2.1 貴金屬（Precious metals） 3
2.1.1 鈀（Palladium） 3
2.1.2 金（Aurum） 3
2.1.3 鉑（Platinum） 4
2.1.4 銠（Rhodium） 4
2.1.5 銥（Iridium） 4
2.1.6 釕（Ruthenium） 5
2.1.7 鋨（Osmium） 5
2.2 生物吸附法 5
2.2.1 金屬之生物吸附介紹 6
2.3 胞外聚合物（Extracellular Polymeric Substances, EPS） 9
2.3.1 胞外多醣體（exopolysaccharides） 9
2.3.2 胞外多醣（exopolysaccharides）金屬吸附機理 10
2.4 生物膜（biofilm） 12
2.4.1 生物膜定義 12
2.4.2 生物膜形成 13
2.5 Ochrobactrum（蒼白桿菌） 14
2.6 Microbacterium（微桿菌） 15
第三章、 實驗架構與材料與方法 16
3.1 實驗架構 16
3.2 實驗材料 17
3.3 實驗方法 24
3.3.1 菌種的篩選與鑑定 24
3.3.2 土壤樣品取得與菌種培養 24
3.3.3 菌種保存 25
3.3.4 分析篩選菌株對貴金屬鈀（Pd）的抗性 25
3.3.5 生物吸附之測定 25
3.3.6 微生物生長曲線（growth curve）之測定 25
3.3.7 菌株染色體DNA（genomic DNA extraction） 26
3.3.8 聚合酶連鎖反應（Polymerase Chain Reaction, PCR） 27
3.3.9 瓊脂凝膠電泳分析（electrophoresis） 28
3.3.10 DNA片段之膠體萃取（gel extraction） 29
3.3.11 Ochrobactrum sp. 71對於貴金屬鈀（Pd）耐受性測試 30
3.3.12 Microbacterium sp. 73對於貴金屬鈀（Pd）耐受性測試 30
3.3.13 生物膜生長測定 31
3.3.14 16S rRNA序列分析 31
3.3.15 胞外多醣體的萃取及純化 31
3.3.16 樣品冷凍乾燥法 32
3.3.17 胞外多醣體吸附重金屬 32
3.3.18 還原醣 33
3.4 HPLC分析胞外多醣體 35
3.4.1 原理及儀器 35
3.4.2 HPLC分析方法與條件 36
3.4.3 醣類定量分析 37
第四章、 結果與討論 44
4.1 菌種的篩選與鑑定 44
4.2 Pseudomonas sp. EJ01抵抗貴金屬能力分析 45
4.3 Ochrobactrum sp. 71抵抗貴金屬能力分析 46
4.4 Microbacterium sp. 73抵抗貴金屬能力分析 47
4.5 菌株對貴金屬鈀與金吸附能力之評估 48
4.6 菌株對貴金屬鈀與金吸附能力之評估 49
4.7 Ochrobactrum sp. 71吸附力貴金屬鈀的能力 50
4.8 Microbacterium sp. 73吸附力貴金屬鈀的能力 51
4.9 Ochrobactrum sp. 71吸附力貴金屬鈀的能力 52
4.10 Microbacterium sp. 73吸附力貴金屬鈀的能力 53
4.11 生物膜的形成能力 54
4.12 胞外多醣體吸附鈀的測量 56
4.13 胞外多醣體吸附鈀的測量 57
4.14 胞外多醣體（EPS）還原糖測定 58
4.15 HPLC之糖類分析 59
4.16 胞外多醣體（EPS）組成與分析 66
4.17 胞外多醣體（EPS）組成與分析 67
第五章、 結論 68
第六章、參考文獻 69

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