黃世佑臺灣大學：化學工程學研究所林家裕Lin, Chia-YuChia-YuLin2007-11-262018-06-282007-11-262018-06-282005http://ntur.lib.ntu.edu.tw//handle/246246/52350本研究係利用Haloferax mediterranei菌株進行250 ml錐形瓶及5 L發酵槽發酵實驗，探討培養基組成以及發酵條件對菌體生長及PHA累積量的影響以尋找大量生產PHA之發酵控制策略。實驗結果發現，鹽類濃度對菌體生長及PHA累積有很大的影響，隨著NaCl、MgSO4、MgCl2濃度增高，菌量也隨之提高，但PHA的累積量卻越低。對於氮源的利用，Haloferax mediterranei對yeast extract利用效果會比利用無機氮有效，並限氮條件並不能促進PHA累積。 在pH影響方面，pH控制在6.5時，菌體生長狀況較佳。在溫度影響方面，當溫度由37 ℃提升到52 ℃時，PHA累積量及PHA含量從0.06 g/L 及0.88 %提升到0.64 g/L及16.6 %，因此提高溫度可加速H. mediterranei累積PHAs。然而，溫度的提升會加速菌體生長及PHA之累積，但對菌量有相當的抑制；在溫度37 ℃時，菌量可達8.8 g/L，溫度在52 ℃時，菌量達5.3 g/L。為使菌體生長不受限制以利於之後大量生產PHA，提出一兩階段溫度控制模式；第一階段控制在37 ℃使菌量大量生長，並在對數生長期中後期在提高溫度至52 ℃，使PHA迅速累積。結果顯示，在此溫度控制策略下，菌量、PHA累積量及PHA含量分別為12.4 g/L、2.44 g/L及20.4 %比溫度控制在52 ℃的結果8.3 g/L、1.14 g/L及15.5 %來的高，因此此溫度控制策略的確可在不抑制菌體生長下，增加PHA的累積量。另外，pH stat批次饋料實驗中，菌量及PHA累積量都有提升，最高分別可達23.23 g/L及4.66 g/L。Effects of various substrates and fermentation conditions, cell growth and accumulation of polyhydroxyalkanoates (PHAs) were investigated using 250 ml shake flasks and a 5 L fermentor. The strain used was Haloferax mediterranei. It was found that the higher the concentrations of NaCl , MgSO4 and MgCl2 , the faster the cell growth and the lower the amount of PHA produced. The cells grew better by using yeast extract as nitrogen source compared to the inorganic nitrogen sources, and PHA accumulation didn’t enhance under nitrogen limitation. The cells grew fastest at pH 6.5. The effect of temperature on cell growth and PHA accumulation was remarkable. PHA production can be stimulated by raising the temperature. The amount of PHA accumulated and PHA content obtained were 0.64 g/L and 16.6 % dry weight, respectively at 52 ℃ which was higher than the 0.06 g/L and 0.88 % obtained at 37 ℃. However, cell mass concentration was lowered as temperature increased. Cell mass concentration at 37 ℃ and 52 ℃ were 8.8 g/L and 5.3 g/L, respectively. To alleviate suppression of cell growth and increase the amount of PHA produced, we proposed a two-step temperature control strategy. At first stage, temperature was controlled at 37 ℃ to enhance cell mass proliferation, and then adjusted to 52 ℃ in the second stage to stimulate the accumulation of PHA. It was found that this strategy was effective to improve the amount of PHA accumulation without suppression of cell growth. Cell mass concentration, amount of PHA produced and PHA content were 12.4 g/L, 2.44 g/L and 20.4 % dry weight, respectively by using this strategy. The results obtained were higher than those under temperature controlled at 52 ℃ throughout. Besides, cell concentration and amount of PHA accumulation can be improved by pH-stat fed-batch operation. The results were 23.23 g/L and 4.66 g/L, respectively.誌謝………………………………………………………….……………I 中文摘要………………………………………………………………...II Abstract…………………………………..……………………………..IV 目錄………………………..………………...…………….……………VI 圖目錄………………………………….……...………………………..XI 表目錄………………………………….……………………..…..…..XIII 第一章 緒論……………………………………………………………..1 1-1前言………………………………………………………………..1 1-2研究目的…………………………………………………………..2 第二章 文獻回顧………………………………………………………..3 2-1生物可分解性高分子簡介………………………………………..3 2-2 PHA之簡介……………………………………………………….3 2-2.1 PHA之結構…………………………………………………4 2-2.2 PHA特性……………………………………………………5 2-3 微生物生產PHA之探討…………………………………………7 2-3.1 利用不同控制策略來生產PHA…………………………...8 2-3.1.1 限氧之控制策略…………………………………...8 2-3.1.2 限氮之控制策略………………………………….10 2-3.1.3 限磷之控制策略…………………….…………....10 2-3.2 利用Haloferax mediterranei耐鹽菌生產PHA………..…11 2-4 PHA應用………………………………………………………...12 第三章 材料與方法………………….……………………………….14 3-1 實驗藥品與試劑………………………………………………...14 3-2 實驗儀器………………………………………………………...16 3-3 實驗流程與方法……………………………………………...…17 3-3.1 菌種來源…………………………………………………..20 3-3.2 培養基……………………………………………………..20 3-3.3 菌種活化與種菌培養……………………………………..22 3-3.4 基質與產物分析方法……………………………………..23 3-3.4.1 葡萄糖濃度測定………………………………….23 3-3.4.2 TKN濃度之測定………………………………….25 3-3.4.3 銨氮濃度之測定………………………………….27 3-3.4.4 菌體乾重之測定………………………………….28 3-3.4.5 PHA之萃取與純化……….………………………28 3-3.4.6 核磁共振儀（NMR）分析………………………29 3-3.4.7 微差熱掃描分析儀（DSC）分析………………..29 3-4 H. mediterranei以葡萄糖為碳源發酵生產PHA……………….29 3-4.1 錐形瓶發酵實驗…………………………………………..29 3-4.1.1 NaHCO3、CaCl2、NaBr有無添加對H. mediterranei生長及PHA累積之影響………………………….30 3-4.1.2 MgCl2及MgSO4添加濃度對H. mediterranei生長及PHA累積之影響…………………………………..30 3-4.1.3 NaCl添加濃度對H. mediterranei生長及PHA累積之影響……………………………………………..31 3-4.1.4 不同氮源對H. mediterranei生長之影響………..31 3-4.1.5 初始NH4Cl添加濃度對H. mediterranei生長之影響…..……………………………………………..31 3-4.1.6 初始yeast extract濃度對H. mediterranei生長及PHA累積之影響…………………………………31 3-4.2 5 L發酵槽發酵實驗……………………………………….32 3-4.2.1 發酵槽操作步驟………………………………….32 3-4.2.2 pH對H. mediterranei生長及PHA累積之影響…33 3-4.2.3 溫度對H. mediterranei生長及PHA累積之影響.34 3-4.2.4 初始葡萄糖濃度對H. mediterranei生長及PHA累積之影響……………………………..…………..34 3-4.2.5 兩段式溫度控制對H. mediterranei生長及PHA累積之影響…………………………………………..34 3-4.2.6 利用pH stat控制策略進行批次饋料發酵………35 第四章 結果與討論……………..……………………………………..36 4-1 錐形瓶發酵實驗………………………………………………...36 4-1.1 NaHCO3、CaCl2、NaBr之添加對H. mediterranei生長及PHA累積之影響…………………………………………..36 4-1.2 MgCl2及MgSO4添加濃度對H. mediterranei生長及PHA累積之影響………………………………………………..38 4-1.3 NaCl添加濃度對H. mediterranei生長及PHA累積之影響…………………………………………………………..41 4-1.4 不同氮源對H. mediterranei生長之影響………………..43 4-1.5 初始氯化銨添加濃度對H. mediterranei生長之影響…..44 4-1.6 初始Yeast extract濃度對H. mediterranei生長及PHA累積之影響……………………………………………………..46 4-2 5 L發酵槽發酵實驗……………………………………………..49 4-2-1 pH對H. mediterranei生長及PHA累積之影響…………49 4-2-2溫度對菌體生長及PHA累積之影響…………………….53 4-2-3初始葡萄糖濃度對H. mediterranei生長及PHA累積之影響..…………………………………………………………57 4-2-4兩段式溫度控制對菌體生長及PHA累積之影響………61 4-2-5利用pH stat控制策略進行批次饋料發酵……………….64 4-3 PHA性質測定…………………………………………..……….67 4-3.1 NMR測定………………………………………………….67 4-3.2 微差熱掃描分析儀（DSC）分析…………………………69 第五章 結論……………………………………………………………70 第六章 參考文獻………………………………………………………73 附錄1 酵素電極反應時之溶氧變化…………………………………..77 附錄2 酵素活性pH值之最佳化………………………………………78 附錄3 葡萄糖濃度標準曲線…………………...……………………...79 附錄4 TKN 濃度標準曲線…………………………………………….80 附錄5 銨氮濃度標準曲線……………………………………………..811058227 bytesapplication/pdfen-USHaloferax mediterranei聚羥基烷酯溫度pH-stat批次饋料Poly-β-hydroxyalkanoatestemperaturepH-stat fed-batchthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/52350/1/ntu-94-R92524040-1.pdf