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研究生:張濬儒
研究生(外文):Jhang, Jyun-Ru
論文名稱:假單胞菌屬生物界面活性劑較適化培養條件之研究
論文名稱(外文):Study on the Optimal Culture Conditions of Pseudomonas Biosurfactant
指導教授:柴浣蘭
指導教授(外文):Chai, Wan-Lan
口試委員:柴浣蘭周錦東何一正傅崇德
口試委員(外文):Chai, Wan-LanChow, Jing-DongHo, Yi-ChengFu, Chung-Te
口試日期:2017-10-27
學位類別:碩士
校院名稱:萬能科技大學
系所名稱:環境工程研究所
學門:工程學門
學類:環境工程學類
論文種類:學術論文
論文出版年:2018
畢業學年度:106
語文別:中文
論文頁數:101
中文關鍵詞:生物界面活性劑假單胞菌鼠李醣脂乳化指數表面張力
外文關鍵詞:biosurfactantPseudomonasrhamnolipidemulsification indexsurface tension
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  隨著工業科技的快速發展,石油已成為現今最重要的石化原料及工業燃料。雖然石油可為人們帶來許多便利,但在其提煉過程、輸送過程及貯存時都可能對自然環境造成污染,而土壤及地下水為最直接的承受體。土壤與地下水整治技術中,生物復育技術是一種具經濟性、對環境較友善且符合自然界機制的方式。技術關鍵在於微生物的效能以及環境的控制,若微生物狀態控制良好,同時提高污染物之生物可及性(Bioavailability),可藉此達到縮短整治時程的目標。因此本研究將把重點放在微生物的培養,嘗試以系統化的方式培養菌種,藉此提升微生物處理油品的能力與整治效果。

  本研究使用之菌種是由南部某柴油污染場址之土壤篩選出,是一株同時具有乳化能力以及清除苯和萘能力的假單胞菌,經鑑定後發現為一株全新的菌種,它被命名為桃園假單胞菌(Pseudomonas taoyuanensis)。此菌種於代表性評估指標試驗中以37 ℃靜置培養120小時,乳化指數可高達73.88 %、OD600大約可達到4、rhamnolipid含量落在1,500至2,000 mg/L之間以及可將表面張力降低至54 dyne/cm。由於乳化試驗操作較簡易,且能夠立即以肉眼觀察到菌種乳化油品的狀況,因此最後以乳化試驗作為本研究之代表性評估指標。

  在較適化控制參數的四個參數中,以轉速200 rpm震盪培養後的微生物濃度及乳化能力都不及靜置培養的表現,且需耗費額外的能源,整體來看震盪培養對於此菌種並沒有太大的幫助。此外,本菌種對高溫的耐受性較差,而在低溫及中溫的環境下培養其乳化能力都相當優異,證明可廣泛應用於台灣的污染場址。而培養過程中溶氧會被迅速消耗殆盡,但其乳化能力並不會受到影響,且就算培養前額外供給氣體,仍無法再提升乳化能力。最後,此菌種培養96小時之後其乳化指數即呈現緩慢上升,於培養120小時達到最高值,在考量培養菌種的時間及成本下,建議未來培養此菌種時培養96小時即可。
  The usage of petrochemical products increases the risk of soil contamination that becomes one of the major environmental problems. Bioremediation is an ecofriendly technology for soil remediation. The effectiveness of strain and regulation of environment are thus critical factors. Although bioremediation is a kind of economic technology to the environment, bioavailability is limited to the effect of remediation. Therefore, the goal of this study is to find the optimal culture conditions of strain to enhance the effects of bioremediation, and achieve the aims of shortening the remediation schedule.

  Pseudomonas taoyuanensis is isolated from the diesel-contaminated soils in southern Taiwan and identified as a novel strain by 16S rDNA sequencing analysis. P. taoyuanensis has the degrading ability for Benzene and Naphthalene and show an excellent emulsifying activity. In the experiment of optimal culture conditions, we investigate the effects on agitation, temperature, aeration and incubation time. The influence of agitation and aeration on the incubation time is not obvious to the growth of P. taoyuanensis. The optimal culture temperature time is 37 ℃ and incubation time is 96 hours. Microbial concentration was estimated by the optical density of the fermentation broth at 600 nm and the OD600 of P. taoyuanensis is approximately 4. Concentration of rhamnolipid produces by P. taoyuanensis was quantified between 1,500 to 2,000 mg/L by the orcinol-sulphuric acid method. P. taoyuanensis can reduce surface tension of water from 72 to 54 dyne/cm and emulsification index against diesel is more than 70 %. This study demonstrated that P. taoyuanensis is less tolerant to 50 ℃ and has the emulsifying activity. The application of P. taoyuanensis is a potential technology in contaminated sites.
摘要 i
Abstract iii
誌謝 iv
目錄 v
表目錄 viii
圖目錄 ix
第一章 前言 1
1.1 研究緣起 1
1.2 研究目的及內容 2
第二章 文獻回顧 3
2.1 土壤性質 3
2.1.1 土壤物理性質 3
2.1.2 土壤化學性質 6
2.2 柴油組成及特性 10
2.2.1 柴油組成 10
2.2.2 柴油特性 11
2.2.3 柴油毒性 12
2.3 界面活性劑 14
2.3.1 界面活性劑之種類 14
2.3.2 化學界面活性劑 16
2.3.3 生物界面活性劑 17
2.3.4 界面活性劑之應用 21
2.4 生物復育技術 24
2.4.1 生物復育技術原理 27
2.4.2 生物復育技術種類 28
2.4.3 生物復育技術之限制因子 32
第三章 研究方法 36
3.1 研究流程 36
3.2 菌種來源 38
3.2.1 菌種分離純化及保存 38
3.2.2 柴油降解潛勢試驗 38
3.2.3 菌種之鑑定 38
3.2.4 菌種培養 39
3.3 菌種特性分析 40
3.3.1 微生物濃度之測定 40
3.3.2 乳化能力之測定 40
3.3.3 表面張力之測定 41
3.3.4 Rhamnolipid含量分析 41
3.4 菌種較適化培養條件試驗 41
3.4.1 代表性評估指標試驗 42
3.4.2 較適化控制參數試驗 42
3.5 實驗設備及操作條件 44
第四章 結果與討論 45
4.1 菌種來源及鑑定結果 45
4.2 代表性評估指標試驗結果 46
4.3 較適化控制參數試驗結果 61
4.3.1 轉速 61
4.3.2 溫度 67
4.3.3 氣體供給 71
4.3.4 時間 82
第五章 結論與建議 89
5.1 結論 89
5.2 建議 90
參考文獻 91
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