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研究生： 廖茂易
Mao-Yi Liao
論文名稱： 探討不同供氧量對Kineosphaera limosa生產PHBV之影響
Study different supply oxygen limitation for production of PHBV in batch culture of Kineosphaera limosa
指導教授： 徐敬衡
Chin-Hang Shu
口試委員:
學位類別： 碩士
Master
系所名稱： 工學院 - 化學工程與材料工程學系
Department of Chemical & Materials Engineering
畢業學年度： 94
語文別： 中文
論文頁數： 119
中文關鍵詞： 聚羥基丁酯-羥基戊酯共聚物 、生物 可分解材料 、革蘭陽性菌
外文關鍵詞： kineosphaera limosa, oxygen, PHBV
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    • 摘 要
    利用微生物系統生產可分解性的材質，來解決全球普遍性的塑膠廢棄物污染問題，是日益重要的課題。由於塑膠所產生的垃圾污染，已造成環境污染上的一大問題，因此生物可分解性塑膠的研究也隨之而起，聚羥基烷polyhydroxyalkanoates (PHAs)為菌體儲存碳源與能量的物質，可以用來當作生物可分解性塑膠的原料。
    Kineosphaera limosa是一株由劉文佐博士實驗室在厭氧-好氧活性汙泥系統中發現且分離出來的兼性氧與革蘭陽性菌，在適當菌體生長的情況下會生成聚羥基丁酯-羥基戊酯共聚物(Poly(3-hydroxybutyrate-co-3-hydroxyvalerate), PHBV)，而NADH為生成PHAs時所需之還原力(Reducing power)，菌體在微氧的情況下有利NADH的生成，本研究探討菌體在不同的攪拌速率下對產物PHBV產出的影響，以及調整此菌株生成不同PHB/PHV比例的策略。
    搖瓶實驗結果指出微供氧量培養下，三種不同單一碳源：甘油、葡萄醣和醋酸鈉測試，以葡萄醣為單一碳源時產物PHBV有較高產量且其PHV佔全部產物的比例為85%。
    發酵槽實驗中，在不同攪拌速率下，其轉速越高下供氧量就越多，在轉速100至200rpm下，體積氧氣傳送係數(kLa)從1.91 hr-1增3.68 hr-1，
    可以發現菌體的比生長速率就有越快趨勢從0.009 hr-1至0.024 hr-1。其轉速越高下供氧量就越多，其產生PHB/PHBV比例也不同，由5%至99%，因此攪拌速率下降會使氧氣質傳下降，造成菌體比生長速率下降而會使PHV產出的比例增加。
    在兩階段不同攪拌速率，由高轉速切換至低轉速下，攪拌速率由200 rpm經66小時發酵後切換為100 rpm發酵，其結果是因為在200 rpm的條件下，菌體生長速度快而在葡萄醣的消耗速度太快下，不易選擇適當的切換時間點；有了之前的經驗改由低轉速切換至高轉速下，攪拌速率由150 rpm經85小時發酵後切換為220 rpm發酵，未切換前PHB/PHBV為0%，而切換後，所產生PHB/PHBV比例也由17%降至13%。比固定低攪拌速率100 rpm~150 rpm所產生PHB比例5%~6%為高。

    Abstract
    Producing the biodegradable material by using mircoorganism system. It is a more important topic to solve the pollution of plastics in the whole world. The environmental pollution is a big problem as a result of the pollution of plastics. For this reason, people start studying biodegradable material. Biodegradable polymers, poly(3-hydroxybutyric) acid (PHB), and a copolymer, poly(3-hydroxybutyric-cohydroxyvaleric) acid [P(HB-co-HV)], are accumulated inside the cells as reserves.
    Kineosphaera limosa is a high-G+C Gram-positive, motile, non-spore-forming coccus capable of accumulating significant amounts of polyhydroxyalkanoate (PHA) was isolated from an inefficient biological phosphorus removal activated sludge reactor by Dr. Liu Wen-Tso. The NADH needs reducing power during producing PHAs. And it is good for the bacteria with the few oxygen to producing the NADH. In this study, I researched about different agitation versus producing PHBV and producing different PHB/PHV ratio by using two stages.
    In the flask experiments, the test of three different single carbon source：glycerol、glucose and sodium acetate, when the glucose is the only single carbon source, the quantity of PHBV output is more and PHV/PHBV ratio is 85%.
    In the fermentation experiments, with different agitation rate, the higher rate will provide more oxygen. The oxygen transfer rate coefficient (kLa) is from 1.91 to 3.68 hr-1，when the agitation rate is from 100 to 200 rpm. With higher agitation rate, the PHB/PHBV ratio is different from 5% to 99%. And lower agitation rate will make the oxygen transfer down, that the specific
    growth rate(μ) is decreasing but the PHV/PHBV ratio is increasing.
    After the agitation rate 200 rpm for 66 hr, the agitation rate was shifted to the agitation rate 100 rpm In two stage different agitation rate. Because the specific growth rate(μ) is fast during agitation rate 200 rpm, it is difficult to look for the suitable shifted point. With prior experience, I started the agitation rate from 150 rpm to 220 rpm. After the agitation rate 150 rpm for 85 hr, the agitation rate was shifted to the agitation rate 220 rpm. Before shifting ,the PHB/PHBV ratio is 0%. After shifting, the PHB/PHBV ratio is decreasing from 17% to 13%. It is higher than PHB/PHBV ratio 5~6% during the single agitation rate 100~150rpm.

    目 錄 目錄………………………………………………………………….....…Ⅰ 表索引……………………………………………………………….....…Ⅵ 圖索引……………………………………………………………….....…Ⅷ 第一章 ﹑緒論…………………………………………..............................1 1.1 研究背景………………………………………………….............1 1.2 研究目的………………………………………………….............5 第二章﹑文獻回顧………………………………………………………..6 2.1 分解性塑膠之簡介……………………….....................................6 2.1.1 分解性塑膠的分類………………………………..................6 2.1.2 生物分解性塑膠的分類…………………..............................8 2.2 微生物的代謝作用..................................................................11 2.2.1 初級 代謝的生合成...........................................................11 2.2.2 次 級 代謝的生合成...........................................................11 2.2.3 微生物生長和產物形成的關係...................................14 2.2.4 微生物發酵動力學-菌種的生長曲線.....................................15 2.3 PHAs之簡介……………………………………............................17 2.3.1 PHAs之發現與發展……………………………………….....17 2.3.2 PHAs之結構與物理化學性質…………………….................19 2.3.3 PHAs之合成代謝路徑….........................................................23 2.3.4 可生產PHAs之菌株...............................................................30 2.3.5 PHAs之生物分解性.................................................................32 2.4 深層液態發酵培養..........................................................................33 2.4.1深層發酵培養的優點................................................................34 2.5 影響發酵的物理化學因子…………………………………..........34 2.5.1 培養基組成…………………………………………...............34 2.5.1.1 碳源…………....................................................................34 2.5.1.2 氮源…………………………………………………........36 2.5.1.3 碳氮比………………………………………………........36 2.5.1.4 無機鹽類……………………………………………........37 2.5.1.5 其他添加物…………………………………………........37 2.5.2 培養條件………………………………………………….......38 2.5.2.1 pH值………………………………………………...........38 2.5.2.2 攪拌速率……………………………………………........38 2.5.2.3 溶氧值……………………………………………............39 2.5.2.4 溫度……………………………………………................40 2.5.2.5 接種量………………………………………....................41 2.5.2.6 不同形式的發酵槽...........................................................42 2.5.2.7 黏度...................................................................................42 2.6 發酵動態行為的數學模式.............................................................43 2.6.1 與生長有關的產物(growth-associated product).....................43 2.6.2 與生長無關的產物(nongrowth-associated product)...............44 2.6.3 混合生長相關的產物(mixed-growth-associated product).....44 2.7 OUR與kLa值的量測.....................................................................45 第三章﹑實驗材料與方法………………………………….....................48 3.1 論文架構........................................................................................48 3.2 實驗材料…………………………………………........................49 3.2.1 實驗菌株……………………………………….....................49 3.2.2 實驗藥品……………………………………….....................51 3.2.3 實驗儀器與設備……………………………..………….......52 3.3 實驗方法………………………………………...........................54 3.3.1 菌株保存…………………………………............................54 3.3.2 菌種之活化............................................................................55 3.3.3 培養基組成及操作條件........................................................55 3.3.3.1 固態平板培養.................................................................55 3.3.3.2 種瓶液態培養.................................................................56 3.3.3.3 搖瓶液態培養..................................................................57 3.3.3.4 發酵槽液態培養...............................................................58 3.3.4 分析方法…………………………………..............................59 3.3.4.1 取樣………………………………...................................59 3.3.4.2 分離菌體(Biomass)及發酵液(Broth)…………...............59 3.3.4.3 還原糖濃度測定(Glucose，S)…………….....................60 3.3.4.4 菌體乾重測定(Biomass，X)………………....................61 3.3.4.5 溶氧值測定(DO)...............................................................61 3.3.4.6 酸鹼值測定(pH)................................................................61 3.3.4.7 PHAs分析…......................................................................62 3.3.4.7.1 PHAs分析流程圖.......................................................62 3.3.4.7.2 PHAs酯化和萃取實驗步驟.......................................63 3.3.4.7.3 GC儀器測試實驗步驟...............................................64 3.3.4.8 kLa值測定.......................................................................67 3.4 各測試方法之標準檢量線……………………..........................67 3.4.1 葡萄醣殘量之標準檢量線……………………...................67 3.4.2 PHB和PHV之標準檢量線…………………….................68 3.3.4.8 kLa值與攪拌速率關係.......................................................69 第四章﹑結果與討論………………………………...................................70 4.1 搖瓶培養實驗……………………………......................................70 4.1.1 不同碳源對Kineosphaera limosa的生長影響.......................70 4.1.2 不同碳氮比對Kineosphaera limosa的生長影響...................72 4.2 攪拌式反應器批次發酵槽培養實驗..............................................75 4.2.1 不同攪拌速率對Kineosphaera limosa生產PHBV的影響...75 4.2.1.1 發酵動力曲線的比較........................................................75 4.2.1.2 發酵時間的比較................................................................81 4.2.1.3 不同攪拌速率對菌體生長的影響....................................81 4.2.1.4 不同攪拌速度對於產物PHBV的影響..............................82 4.2.2 兩階段不同攪拌速率對Kineosphaera limosa生產PHBV的影響..............................................................................................84 4.2.2 1 發酵動力曲線的比較........................................................84 4.2.2.2 兩階段不同攪拌速率對生成PHBV的影響....................88 第五章﹑結論與建議………………………...............................................90 5.1 結論..................................................................................................90 5.2 建議..................................................................................................92 第六章﹑參考文獻…………………………...............................................93

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