臺灣博碩士論文加值系統

本研究之目的為從不同環境中採集含油脂之廢水，從中分離篩選出具有油脂分解能力之菌株。首先利用特定培養基進行培養篩選，一共分離出32株具分泌脂肪酵素能力的細菌菌株。利用培養在特殊培養基上所生成白色沉澱環大小來初步判斷其分解能力差別，進行兩次篩選後總共挑出15株菌進行分類鑑定。其中能力最強者為編號S-6，次強者為編號H-4；第三強者為編號G-3，進一步將該菌株分離純化及菌種鑑定，根據16S rDNA sequencing序列結果得知菌名分別依序為Serratia marcescens；Aeromonas hydrophila ； Aeromonas veronii strain。
本研究將所分離菌株與自行由菌種中心購買已知具油脂分解能力之純菌株Azospirillum brasilense 14325進行比較，探討菌株之基本性質及進行脂肪酵素活性測試。Serratia marcescens在固態培養基上白色沉澱環約0.6 cm；Azospirillum brasilense 14325白色沉澱環形成速度緩慢，形狀多僅只呈現點狀分泌。Serratia marcescens在液態培養時所測得之脂肪酵素活性為(71 U)；Azospirillum brasilense 14235之脂肪酵素活性為(0 U)。
進一步探討Serratia marcescens在不同生長條件下之脂肪酵素分解變化，結果顯示此菌株培養在6% Tween 80濃度下，其脂肪酵素活性(506 U)為最佳。pH 值不同時，Serratia marcescens於pH 8 脂肪酵素活性最佳 (667 U)。溫度不同時，於30 ℃培養下之脂肪酵素活性最佳(667 U)。最後將6% Tween 80改用6% 沙拉油取代，發現其在第3天、第5天、第7天之油脂降解率分別為7.43%、14.56%、29.41%。推測將Serratia marcescens應用到廢食用油降解時，將可以得到不錯的降解效果。

The objective of this study was to isolate and identify oil-degrading bacteria from different wastewater environments. First of all, a selective media was used as the screening tool for lipase-secreting bacteria, and a total of 32 strains were isolated. The lipase activity of these strains was identified by detecting the clear zone surrounding the bacterial colony when incubated on the specific media plate. After two separated screening processes, 15 strains were selected for detailed study. The strain which have the strongest ability of lipase production was numbered as strain S-6, followed by strain H-4 and G-3. Based on the 16S rDNA sequence analysis results, these strains were identified as Serratia marcescens, Aeromonas hydrophila and Aeromonas veronii, respectively.
In this study, the growth characteristics and lipase activity of the isolated strains, were compared with Azospirillum brasilense 14325 (purchased from Bioresource Culture Research Center), one of the lipolytic strains reported in the reference. The size of clear zone produced by Serratia marcescens on the solid medium was about 0.6 cm. But the clear zone was formed slowly by Azospirillum brasilense 14325, and had only pinpoint-like shape of secretion observed. Llipase activity of Serratia marcescens and Azospirillum brasilense 14235 when cultured in liquid medium were (71 U) and (0 U), respectively.
Further examination of the lipase activity of Serratia marcescens under different growth conditions showed that the best lipase activity (506 U) could be obtained when this strain was incubated under a concentration of 6% Tween 80, pH8 and 30oC. Finally, when the use of 6% Tween 80 was replaced by 6% salad oil, the oil decomposing rate were 7.43% (3 days), 14.56% (5 days), 29.41% (7 days). We speculated that Serratia marcescens strain may have good degradation effects when applied to the processing of waste edible oil.

封面
口試
致謝
中文摘要 i
Abstract ii
目錄 iii
表目錄 vi
圖目錄 vii
第一章 前言 1
第一節 研究緣起 1
第二節 研究目的 2
第二章 文獻回顧 3
第一節 油脂的特性 3
第二節 油脂廢水的來源 4
第三節 油脂廢水的處理 5
第四節 脂肪酵素之介紹 7
一、 脂肪酵素 7
二、 脂肪酵素生物特性 8
三、 微生物中脂肪酵素來源 9
四、 脂肪酵素反應形式 9
五、 脂肪酵素催化機制 10
六、 脂肪酵素與酯解酵素之差異 11
第五節 脂肪酵素之應用 13
第六節 界面活性劑簡介 16
一、 界面活性劑的分類 16
二、 HLB 值( Hydrophilic-Lipophilic Balance ) 18
第七節 Tween80之簡介 19
一、 利用Tween80於脂肪酵素之相關研究 19
二、 Tween 80介紹 20
第八節 具有脂肪酵素之微生物 22
一、 微生物的利用 24
二、 微生物的生物降解反應 27
第三章 實驗材料與方法 30
第一節 實驗架構 30
第二節 儀器設備 31
第三節 脂肪分解菌篩選方法 32
一、 菌種來源 32
二、 菌種分離純化 32
三、 培養基 33
四、 篩選具有脂肪酵素活性菌株 35
五、 菌落特性介紹 35
六、 菌株保存 35
第四節 菌種16S rDNA鑑定操作步驟 36
一、 核酸萃取 36
二、 聚合酶連鎖反應(PCR) 37
三、 聚合酶連鎖反應(PCR)條件 38
四、 電泳分析 38
五、 變性梯度凝膠電泳(DGGE)分析 39
六、 DNA序列分析 40
第五節 脂肪酵素活性分析 41
第六節 脂肪酸鑑別試驗及含量分析 43
一、 菌株在液態培養基下對Tween 80之利用實驗 43
二、 氣相層析儀分析條件 43
三、 貯存標準液配製 44
四、 檢液配製 45
第七節 菌株最適生長條件實驗 46
一、 Tween80濃度對脂肪酵素影響 46
二、 pH值對脂肪酵素影響 46
三、 溫度對脂肪酵素影響 47
第八節 菌株在液態培養下對沙拉油利用實驗 48
一、 萃取重量法 48
第四章 結果與討論 49
第一節 脂肪分解菌篩選 49
第二節 分離菌株特性觀察 50
第三節 菌株定序結果 55
第四節 本研究所得之脂肪分解菌特性整理 59
第五節 Serratia marcescens、Azospirillum brasilense 14325比較 62
一、 脂肪分解菌固態培養基比較 62
二、 脂肪分解菌液態培養基比較 63
三、 九株油脂分解菌之Lipase酵素活性分析結果 65
第六節 探討Serratia marcescens最適生長條件之脂肪酵素變化 67
一、 Serratia marcescens之介紹 67
二、 Tween80濃度對脂肪酵素影響 69
三、 pH值對脂肪酵素影響 71
四、 溫度對脂肪酵素影響 73
第七節 菌株對沙拉油之油脂降解結果 75
第八節 Tween80對菌株之脂肪酸分析結果 76
第五章 結論與建議 80
第一節 結論 80
第二節 建議 82
參考文獻 83
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二、 英文文獻 85

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