聚羥基丁酯(PHB)具有良好的生物降解性、生物相容性及熱塑性等特質，可用來取代傳統高分子材料，避免造成環境的負擔。本研究主要探討放線菌Streptomyces sp. 77T-4對PHB薄膜的分解行為和機制；另外，藉由製備PHB/PVAc和PHB/Biomax摻合薄膜，比較其放線菌Streptomyces sp. 77T-4分解行為的差異。利用溶劑揮發法和融熔熱壓法分別製備表面多孔性和緻密性的PHB薄膜，將所製備的薄膜，經由菌體分解後，由SEM、DSC、GPC、重量損失分析及表面接觸角等測量分解過程中的變化。經由SEM觀察結果，放線菌Streptomyces sp. 77T-4會分泌PHB分解酵素來侵蝕材料的表面，雖然PHB/PVAc和PHB/Biomax摻合薄膜表面皆有分解的現象，但依然可分辨各自為完全相容的摻合系統和不相容的摻合系統。而且發現PHB材料的表面積越大，菌體接觸到高分子的機率越多，而分泌PHB分解酵素的時間會提早，材料的分解程度也會增加；若再利用PHB懸浮液誘導放線菌Streptomyces sp. 77T-4後，可提升材料分解的效率。並且依據DSC和GPC的測試結果，不同分解時間下PHB薄膜的結晶度和分子量，不會隨著分解時間的改變而變化，這是由於PHB分解酵素分解PHB高分子鏈的速度比酵素擴散進入材料的速度還要快速的原因。依據上述結果，本實驗推擬出放線菌Streptomyces sp. 77T-4分解PHB薄膜之機制為 – 初期，菌體會逐漸適應PHB高分子，而活化PHB分解酵素合成機制。之後，則會開始大量的的生產PHB分解酵素，使酵素經擴散作用進入PHB薄膜，造成薄膜表面產生均勻的孔洞，而釋放寡聚物(oliomer)或是羥基丁酸單體，則可作為 Streptomyces sp. 77T-4之營養源而被吸收。

Poly(3-hydroxybutyrate)(PHB) has many good characteristics, like biodegradable, biocompatiblity and plasticity, etc, it’s could be use to substitute for the traditional polymeric materials and avoid to be a  environmental burden. This research mainly discusses the degradation behavior and mechanism of Streptomyces sp. 77T-4 on PHB film. Moreover, respectively make the PHB/PVAc and PHB/Biomax blend films, compared with the difference of their degradation behaviors with Streptomyces sp. 77T-4. Using the solution-evaporation and thermal pressurization to make the films with respectively the surface of porous and dense, and after the degradation by bacteria, exact the change between original and final one by SEM, DSC, GPC, thickness-loss analysis and weight-loss analysis. 
       As the result of the SEM, we found that the PHB degradation enzyme Streptomyces sp. 77T-4 secreted can erosion the material surface. Although the surface of PHB/PVAc and PHB/Biomax blend films both   have the phenomenon of erosion, but it still could be differentiate to the two different system of miscible system and immiscible system.
        And as the results of the degradation analyses by Streptomyces sp. 77T-4, the bigger of the surface area of PHB material, the more probability of the bacterium touch polymers, and the faster the PHB degradation enzyme secreted, and then the extent of material degradation would be increased. If using PHB suspension to induces Streptomyces sp.  77T-4 to secrete the degradation enzyme, may promote the efficiency of the degradation of PHB film.  And by the tests of the DSC and GPC, we found that under the different degradation time, the crystallinity and the molecular weight of the PHB film will not change, because of the speed of the PHB degradation enzyme degraded the PHB polymer chain is faster than the speed of the enzyme diffused into the material. 
       Based on the above results, the progress had extrapolated the degradation mechanism of PHB film by Streptomyces sp. 77T-4 to be – the initial period, 77T-4 would adapt the PHB high polymer gradually, and activate the synthesis mechanism of the PHB degradation enzyme. Afterward, 77T-4 would massively product PHB decomposition enzyme, and the enzyme entered into the PHB film through diffusion to make the even pores on the film surface, and released oligomers or the hydroxy-butyric acid monomer which may be nutrition of source and absorb by the 77T-4.

摘要…………………………………………………………………………..i
目錄	v
第一章     緒論	1
1.1 研究背景	1
1.2 研究動機與目的	3
第二章     文獻回顧與理論基礎	4
2.1 生物可分解高分子(Biodegradable polymers)	4
2.2 聚羥基丁酯 ((Poly(3-hydroxybutyrate), PHB)	8
2.3 高分子聚摻合體(Blends)	11
2.3.1 聚摻合體之製備方式	11
2.3.2 聚摻合物之相容性	12
2.4 PHAs聚摻合物系統	15
2.5 放線菌(Actinomycetes)	18
2.5.1 外觀特徵	18
2.5.2 培養特徵	20
2.5.3 重要種屬	21
2.6 生長曲線(Growth curve)	23
2.7 生物退化(Biodeterioration)	25
2.8 PHAs的微生物酵素分解	30
第三章     實驗方法	35
3.1 實驗藥品	35
3.2 實驗設備與分析儀器	39
3.3 實驗步驟	42
3.3.1 PHB高分子之純化	42
3.3.2 利用溶劑揮發法製備孔洞型PHB薄膜	42
3.3.3 利用熔融熱壓法製備緻密型PHB薄膜	43
3.3.4 PHB懸浮液之製備	43
3.3.5利用溶劑揮發法製備PHB/PVAc與PHB/Biomax摻合薄膜	44
3.3.6 液態培養基 Urea-Fructose medium 之製備	44
3.3.7 基礎培養基(Basal medium)之製備[48]	45
3.3.8 Streptomyces sp. 77T-4之培養	46
3.3.9 Streptomyces sp. 77T-4分解PHB薄膜	47
3.3.10 Streptomyces sp. 77T-4分解PHB懸浮液/PHB薄膜	47
3.3.11 經PHB懸浮液預培養之Streptomyces sp. 77T-4分解PHB薄膜	48
3.3.12 Streptomyces sp. 77T-4之PHB分解酵素活性測試	48
3.3.13 分批式Streptomyces sp. 77T-4分解PHB薄膜	49
3.3.14 PHB薄膜孔隙度測試	49
3.4 實驗分析	51
3.4.1 結構分析	51
3.4.2 形態觀察	52
3.4.3 熱穩定性測試	52
3.4.4 熱轉移溫度及結晶度	52
3.4.5 分子量及分子量分佈	53
3.4.6 結晶構造分析	53
3.4.7 表面接觸角分析	54
3.5 實驗流程	55
第四章     結果與討論	56
4.1 PHB高分子材料之型態與性質分析	56
4.1.1 PHB高分子之純化分析	56
4.1.2 PHB懸浮液之粒徑分析與探討	60
4.2 PHB薄膜之形態探討與性質分析	62
4.2.1 sPHB與mPHB薄膜之結構分析	62
4.2.2 sPHB與mPHB薄膜之形態觀察	63
4.2.3 sPHB和mPHB薄膜之結晶形態	64
4.3 Streptomyces sp. 77T-4基本特性分析	68
4.4 Streptomyces sp. 77T-4直接分解PHB薄膜之性質及形態探討	72
4.4.1 Streptomyces sp. 77T-4直接分解PHB薄膜之酵素活性分析	72
4.4.2 Streptomyces sp. 77T-4直接分解PHB薄膜之重量分析	75
4.4.3 Streptomyces sp. 77T-4直接分解PHB薄膜之形態觀察	78
4.4.4 Streptomyces sp. 77T-4直接分解PHB薄膜之厚度分析	84
4.5 探討添加PHB懸浮液之Streptomyces sp. 77T-4分解PHB薄膜行為	87
4.6 分批式Streptomyces sp. 77T-4分解PHB薄膜	90
4.6.1 Streptomyces sp. 77T-4分解PHB薄膜之形態觀察	90
4.6.2 Streptomyces sp. 77T-4分解PHB薄膜之孔隙度分析	94
4.6.3 Streptomyces sp. 77T-4分解PHB薄膜之接觸角分析	95
4.6.4 Streptomyces sp. 77T-4分解PHB薄膜之熱轉移性質測試	96
4.6.5 Streptomyces sp. 77T-4分解PHB薄膜之分子量與分子量分佈	99
4.7 Streptomyces sp. 77T-4分解PHB/PVAc和PHB/Biomax摻合薄膜	102
4.7.1 PHB/PVAc和PHB/Biomax摻合薄膜的相容性	102
4.7.2 Streptomyces sp. 77T-4分解PHB摻合薄膜之重量分析	104
4.7.3 Streptomyces sp. 77T-4分解PHB摻合薄膜之型態觀察	106
4.7.4 Streptomyces sp. 77T-4分解PHB摻合薄膜之結晶型態	108
4.8 放線菌Streptomyces sp. 77T-4分解PHB薄膜的分解機制和模型	111
第五章     結論	115
第六章      參考文獻	117
圖目錄
圖2- 1 生物可分解高分子的分解作用[4]	5
圖2- 2 聚乙烯醇(PVA)的分解機構[5]	6
圖2- 3聚羥基烷酯(PHAs)之結構	8
圖2- 4 DSC圖中玻璃轉移溫度對相容性的關係	14
圖2- 5 Poly(vinyl butyral)之結構	16
圖2- 6 放線菌的菌絲體之示意圖	19
圖2- 7 典型之細菌生長曲線圖	24
圖2- 8 聚乳酸於酸性條件下的水解機制[34]	28
圖2- 9 酵素分解聚酯類高分子的反應機構 [41]	31
圖2- 10 PHB薄膜的分解的兩種型態[42]	32
圖2- 11 分解前後的P(3HB-5%3HV)薄膜SEM圖和AFM圖	33

圖3- 1實驗架構	55

圖4- 1 利用熱重損失所測purified PHB和unpurified PHB的熱裂解圖	57
圖4- 2 利用熱重損失所測purified PHB和unpurified PHB的一次微分熱裂解曲線	57
圖4- 3 Purified PHB的1H-NMR圖	59
圖4- 4 Purified PHB的13C-NMR圖	59
圖4- 5 PHB懸浮液粒徑分佈	61
圖4- 6 sPHB和mPHB的ATR-FTIR圖	62
圖4- 7不同製備方式所獲得的PHB薄膜表面的SEM圖	64
圖4- 8 sPHB、mPHB和mPHB-2的XRD圖	65
圖4- 9 sPHB、mPHB和mPHB-quenching的DSC圖(第一次升溫)	66
圖4- 10 sPHB、mPHB和mPHB-quenching的DSC圖(第二次升溫)	67
圖4- 11放線菌77T-4培養於固培養基Urea-Fructose agar	68
圖4- 12放線菌Streptomyces sp.77T-4 生長曲線	69
圖4- 13Streptomyces sp.77T-4分解PHB之透明環(Clear Zone)	70
圖4- 14放線菌Streptomyces sp.77T-4 於不同溫度下對PHB的分解能力	71
圖4- 15 sPHB薄膜經過不同的分解時間(45°C、100rpm)下，取出的上清液與PHB懸浮液經過作用後的混濁度(OD650)變化曲線	73
圖4- 16 sPHB薄膜經過不同的分解時間(45°C、100rpm)下，取出的上清液與PHB懸浮液經過作用後的酵素分解速率變化圖	74
圖4- 17 sPHB薄膜經過不同的分解時間(45°C、100rpm)下的蛋白質濃度變化圖	74
圖4- 18 連續式PHB薄膜之重量損失和時間的關係圖	76
圖4- 19連續式PHB薄膜之殘留重量百分比和時間的關係圖	77
圖4- 20 不同分解時間下PHB薄膜之外型變化圖	78
圖4- 21sPHB薄膜之分解前和分解14小時的表面SEM圖	80
圖4- 22 mPHB薄膜之分解前和分解74小時的表面SEM圖	81
圖4- 23 sPHB薄膜之分解前和分解14小時的截面SEM圖	82
圖4- 24 mPHB薄膜之分解前和分解74小時的截面SEM圖	83
圖4- 25 連續式PHB薄膜之厚度損失和時間的關係圖，sPHB為溶劑法製備的薄膜；mPHB為熔融法製備的薄膜	85
圖4- 26 連續式PHB薄膜之殘留厚度百分比和時間的關係圖，sPHB為溶劑法製備的薄膜；mPHB為熔融法製備的薄膜	85
圖4- 27添加PHB懸浮液之sPHB薄膜重量損失和時間的關係圖	89
圖4- 28添加PHB懸浮液之sPHB薄膜殘留重量百分比和時間的關係圖	89
圖4- 29不同分解時間的sPHB薄膜的SEM圖(500×)	91
圖4- 30不同分解時間的sPHB薄膜的SEM圖(×1000)	92
圖4- 31不同分解時間的sPHB薄膜的SEM圖(2500×)	93
圖4- 32不同分解時間下sPHB薄膜的孔隙度變化	94
圖4- 33不同分解時間下sPHB薄膜的接觸角變化	95
圖4- 34不同分解時間下sPHB薄膜的DSC圖 (第一次升溫)	97
圖4- 35不同分解時間下sPHB薄膜的DSC圖 (第二次升溫)	97
圖4- 36 不同分解時間下mPHB薄膜的DSC圖 (第一次升溫)	98
圖4- 37不同分解時間下mPHB薄膜的DSC圖 (第二次升溫)	98
圖4- 38不同分解時間sPHB薄膜的分子量和結晶度變化圖	101
圖4- 39 PHB摻合薄膜之DSC圖 (第二次升溫)	103
圖4- 40 PHB摻合薄膜之殘留重量百分比和 時間的關係圖	105
圖4- 41 sPHB/PVAc摻合薄膜之分解前和分解16小時後的表面SEM圖	107
圖4- 42 sPHB/Biomax摻合薄膜之分解前和分解16小時的表面SEM圖	108
圖4- 43 PHB/PVAc摻合薄膜之DSC圖 (第一次升溫)	110
圖4- 44 PHB/PVAc摻合薄膜之DSC圖 (第二次升溫)	110
圖4- 45  放線菌Streptomyces sp. 77T-4分解PHB薄膜之機制模型	114

 
表目錄
表2- 1 PHB 與聚丙烯於25°C下的物理性質與機械性質之比較 [8]	9
表2- 2 微生物以及所對應的生物可分解材料[28]	26

表3- 1 Urea-Fructose medium 之組成	45
表3- 2 基礎培養基之組成	46

表4- 1 Prified PHB之1H-NMR及13C-NMR圖特性吸收峰	58
表4- 2 sPHB、mPHB、mPHB-1和mPHB-2的熱轉移溫度、熔解熱及結晶度	67
表4- 3 sPHB和mPHB薄膜的起始重量損失時間(onset weight-loss time)和重量損失速率(weight-loss rate)	77
表4- 4 PHB薄膜的厚度損失百分比和重量損失百分比	86
表4- 5 添加PHB懸浮液分解薄膜的起始重量損失時間(onset weight-loss time)和重鏈損失速率(weight-loss rate)	88
表4- 6 不同分解時間sPHB薄膜的熱轉移溫度、熔解熱及結晶度	99
表4- 7 不同分解時間sPHB薄膜的數目平均分子量(Mn)、重量平均分子量(Mw)和分子量分佈(PDI)	100
表4- 8 sPHB摻合薄膜的熱轉移溫度、熔解熱及結晶度	103
表4- 9 sPHB/PVAc和sPHB/Biomax摻合薄膜的起始重量損失時間(onset weight-loss time)和重量損失速率(weight-loss rate)	105
表4- 10 PHB/PVAc摻合薄膜的熱轉移溫度、熔解熱及結晶度	109

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