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研究生:洪昭尹
研究生(外文):Jhao-Yin Hung
論文名稱:生產聚羥基酯類發酵策略應用於廢水處理之可行性研究
論文名稱(外文):Feasibility study on applications of fermentation strategy for polyhydroxyalkanoate production to wastewater treatment
指導教授:陳博彥陳博彥引用關係
指導教授(外文):Bor-Yann Chen
口試委員:魏毓宏陳姍玗藍祺偉薛仲娟
口試委員(外文):Yu-Hong WeiShan-Yu ChenChi-Wei LanChung-Chuan Hsueh
口試日期:2013-05-27
學位類別:碩士
校院名稱:國立宜蘭大學
系所名稱:化學工程與材料工程學系碩士班
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:127
中文關鍵詞:深層海水聚羥基酸酯生產生物可分解高分子兩階段式發酵培養
外文關鍵詞:Deep seawaterPolyhydroxyalkanoatebiodegradable polymertwo-stage fermentation
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基於對環境友善綠色科技材料之永續應用,以微生物生產生物可分解性高分子Polyhydroxyalkanoate (PHA)為替代塑料現已成為環境保護之必然趨勢。在海水以及淡水分別進行篩選功能性菌株,研究以自台灣各地之水樣和土樣來進行本土微生物資源開發,以評估用來生產具生物分解性高分子之聚羥基酸酯作為替代塑料之可行性。首先以不同地區所篩選出本土脫色菌種Microvirgula aerodenitrificans SH7b、Rahnella aquatilis DX2b、Pseudomonas sp. SH98及Acinetobacter guillouiae AX9以及Aeromonas hydrophila NIU01, YTl1, KB23及Klebsiella pneumoniae ZMd31,在以蘇丹黑染色法作為初步生產菌株篩選下,以不同碳源作為可行性碳源進行批次發酵培養,分別就菌體生長與聚羥基丁酸酯產量之高低來評估其最適化碳源濃度。發酵槽以第二階段為刺激PHAs生產,以限制氮源培養液(C/N=60)添加相同濃度之椰子油來作為刺激微生物生產PHB之策略,以限制營養源等方式來有效刺激微生物生產目標產物。研究指出以,二階段式發酵雖然需多更換一次基質,卻可更有效促進菌體生長,此發酵策略約可以減少2/3倍的工作時間與操作成本,並提高乾菌重(16.8 g L-1)、PHB含量(62.1 wt%)與濃度(10.4 g L-1),另外在348小時HHx含量約可達到(5.33wt%),此時HHx占全體PHAs更達到8.58%。以RR141染料脫色原廢液培養時,發現其中又以NIU01耐毒性最好,A. hydrophila NIU01於100% RR141染料脫色廢液發酵生產的乾菌重可達3.03 g L-1、PHB含量52.5 wt%與濃度1.67 g L-1,另外PHHx含量的可達到2.35wt%,此時PHHx占全體PHAs的4.23%。在以海洋深層水餵養得到鳳螺腔腸進行篩選之菌體,可得到純菌MSG11與MSu11,並予以鑑定。研究並以MSG11搭配中心混成設計找出MSG11耐鹽菌生產PHB之最佳條件,以利後續工程應用。
For sustainable applications of eco-friendly materials, using microbes to produce biodegradable polymer- polyhydroxyalkanoate (PHA) via green technology to replace plastics seems to be inevitable. Using freshwater and marine-based samples, we explored indigenous microbial resources to isolate functioning bacteria. Meanwhile, feasibility assessment upon capabilities of producing PHAs in place of popularly-used plastics was also implemented. First, we adopted naturally-occurring dye-decolorizers Microvirgula aerodentirificans SH7b, Rahnella aquatis DX2b,Pseudomonas sp. SH98, Acinetobacter guillouiae AX9, Aeromonas hydrophila NIU01, YTl1, KB23, Kelbsiella pneumoniae ZMd31 as model microbes to screen the viability of PHA accumulation via Sudan-black staining. Then, bactch cultures with various carbon sources were carried out to determine optimal carbon concentrations through comparative evaluation of microbial growth and PHA production. In addition, with identical concentration of coconut oil, nitrogen-limiting strategy (C/N ratio of 60) was conducted to stimulate PHA production via second stage fermentation. The findings also indicated that although two-stage fermentation required one more step to replace nutrient substrate during production, it effectively enhanced microbial growth and polyhydroxybutyrate (PHB) expression. This significantly reduced ca. 2/3 operation time and cost, leading to achieve dry cell weight (DCW) 16.8 g L-1, PHB 62.1 wt% and PHB concentration 10.4 g L-1, HHx content 5.33 wt% (ca. 8.58% total PHAs). In cultures with reactive red 141 (RR141)- decolorizing waste broth, strain NIU01 evidently showed the highest toxicity resistance. In particular, using RR141-decolorizing broth as culture media NIU01 could still obtain DCW 3.03 g L-1, PHB content 52.5 wt% and PHHx 2.35 wt% (ca. total PHAs 4.23 %) for materials recycling. Moreover, isolation of gut microbes in deep seawater-cultured Babylonia areolata obtained strains MSG11 and MSu11 as dye-decolorizing bacteria. This study also proposed central composite design of experiments to determine optimal conditions for PHB production of strain MSG11.
摘要 I
誌謝 III
目錄 IV
圖目錄 VIII
表目錄 X
第一章 緒論 1
1.1 前言 1
1.2 研究架構 8
第二章 文獻探討與回顧 9
2.1 生物可分解性塑膠 9
2.1.1 生物可分解性塑膠 9
2.1.2 聚羥基酯類(Polyhydroxyalkanoates, PHAs) 14
2.1.3 PHAs萃取純化分離 22
2.2 環境本土微生物介紹 25
2.2.1 本土淡水微生物來源及介紹 25
2.2.2 耐鹽微生物來源及介紹 28
2.3 實驗設計法 30
2.3.1 實驗設計(Design of Experiment,DOE) 30
2.3.2 實驗設計法 32
第三章 材料與方法
3.1 實驗藥品 35
3.2 儀器設備與實驗器材 37
3.3 微生物培養 39
3.4 培養液 39
3.5 微量元素 40
3.6 蘇丹黑(Sudan Black B)染色法 42
3.7 搖瓶發酵培養 43
3.8 發酵槽操作 44
3.8.1 批次發酵 44
3.8.2 兩階段批次發酵 44
3.9 氣相層析儀(Gas Chromatograph, GC)定量PHB 46
3.9.1 樣品前處理 46
3.9.2 氣相層析儀條件設定 47
3.9.3 PHB檢量線與含量及濃度之計算方法 47
3.10 SDS-次氯酸鈉萃取法 (SDS-NaClO Extraction) 48
3.11 象牙鳳螺腔腸之微生物篩選 49
3.11.1 篩選培養液與碳源 49
3.11.2 PHB生產連續馴養 49
3.11.3 微生物分離純化 50
3.11.4 蛋白質電泳(Sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS-PAGE) 50
3.11.5 親緣樹菌種鑑定 53
3.12 材料性質測試 53
3.12.1 DSC熱分析相變化測試 53
3.12.2 拉力測試 54
3.12.3 凝膠滲透層析儀(GPC) 54
第四章 結果與討論 55
4.1 篩選具可生產PHB之本土性污染物處理微生物 55
4.1.1 PHB生產可行性分析 55
4.1.2 PHB生產定量分析 56
4.2 批次PHB生產之影響 62
4.2.1 最佳碳氮莫耳比值決定 62
4.3 響應迴面法I(Response-Surface Method;RSM) 64
4.3.1 單體格子設計(Simplex-lattice designs) 64
4.3.2 單體格子設計之數據比較 66
4.4 限制營養源決定 71
4.4.1 最佳營養源限制碳氮、碳磷莫耳比值 71
4.5 發酵槽生產PHB之探討 73
4.5.1 批次發酵 73
4.5.2 兩階段式發酵策略可行性應用 77
4.6 鳳螺腔腸菌相分析 85
4.6.1 鳳螺腔腸篩菌流程示意圖 85
4.6.2 可利用生產PHB之碳源篩選 87
4.6.3 鳳螺腔腸馴養與純化 89
4.6.4 蛋白質電泳初步分析(SDS-PAGE) 92
4.6.4 鳳螺腔腸微生物種鑑定 94
4.7 響應曲面法I 98
4.7.1 中心混成設計(Central Composite Design,CCD) 98
4.8 材料實驗 101
4.8.1 中心混成設計(Central Composite Design,CCD) 101
4.8.2 凝膠滲透層析 103
4.9 廢水實驗 104
4.9.1 RR141染整廢水實驗 104
第五章 結論及未來展望 106
第六章 參考文獻 109
附錄 113
自述 126
著作發表 126

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