臺灣博碩士論文加值系統

本研究用來生產PHB之菌株（海洋弧菌Vibrio sp. M11）篩選自本土之海洋環境。根據之前研究結果顯示，Vibrio sp. M11發酵合成之生物高分子種類主要為聚(3-羥基)丁酯（poly(3-hydroxy butyrates), P(3HB)）。本研究將著重於Vibrio sp. M11發酵培養基之最適化及策略之探討，在培養基最適化方面，分別進行碳、氮、NaCl之探討，經由實驗證實，Vibrio sp. M11發酵合成P(3HB)最佳之培養基，其組成如下：10 g/L Glycerol、2 g/L Yeast extract、2.5 g/L Trypton、28 g/L NaCl，此培養基命名為MGYT，而以MGYT進行搖瓶發酵，其PHAs之含量與產量分別為39.58 %與1.9 g/L，相較於未最適化之培養基比較，P(3HB)之含量提升約46 %。研究進一步顯示，額外添加離子，亦將影響PHB產量之代謝合成。研究證實於最適化之MGYT培養基同時加Na2HPO4、KH2PO4、(NH4)2HPO4 與MgSO4四種離子後，其菌體乾重由4.81 g/L上升至8.14 g/L，而PHB由原來39.58 %下降至1.58 %，且pH値由4.5上升至6.8，同時由殘餘Glycerol的分析得知，加入離子之搖瓶培養，碳源於12小時培養後，消耗殆盡。因此離子之添加使Vibrio sp. M11有效利用碳源，增加菌體之生長，使PHB無法有效累積。為了進一步探討其原因，因此個別以去除單一離子與單一離子之添加進行探討，研究發現，Na2HPO4添加後為造成此現象之主因。由於上述結果得知，當培養基之營養源不足時，是較不易於PHB之生成，因此利用發酵策略克服此問題。以饋料批次培養時，(4:1)之碳、氮比例當Feeding soultions，每12小時饋料一次，經48小時培養後，可得最佳之發酵結果，其CDW、PHB之含量與PHB之產量分別為20.13 g/L、38.25 %、7.16 g/L，PHB之產量是MGYTT的7倍，因此結合較適化的培養基與可行發酵策略，為具有量產PHB之潛力。

In this study we used Vibrio sp. M11 to produce biodegradable polymer, Polyhydroxybutyrate(PHB). In order to enhance PHB production, some investigations including medium optimization, growth conditions and fermentative strategy were carried out. The optimal semi-defined MGYT medium was determined as follows: 10 g/L glycerol, 2 g/L yeast extract, 2.5 g/L tryptone, 28 g/L NaCl through the flask test with various concentrations to each component, and PHB content and PHB concentration were 39.58 % and 1.9 g/L, respectively. The research demonstrated that PHB production, metabolism and synthesis would be effected due to elements addition. After adding(Na2HPO4,KH2PO4, (NH4 )2HPO4, MgSO4 ∙7H2O) elements in optimal semi-defined MGYT medium, CDW would be improved from 4.81 g/L to 8.14 g/L, PHB content reduces by 39.58 % to 1.58 %, and the pH value increases by 4.5 to 6.8. It was speculated that elements induced intracelluar depolymerase expression and PHB granules would be degraded for cell growth. Meanwhile, by residual glycerol analysis, glycerol can be to tally consumpted in 12 hr incubation. Therefore elements addition can easily enchance Vibrio sp. M11 to uptake carbon source and increases cell growth, but not easy to produce PHB. In order to know effects of the ion, therefore individually removes sole ion and increase sole ion carries on the discussion. By results mentioned above we found that when nutrition of culture medium is insufficiency, it is not easy for PHB production. Therefore this research wants to solve this problem using the fermentation strategy. For fed-batch fermentation strategy, the use of feeding medium with the C/N ratio at 4:1 lead CDW to 20.13 g/L and PHB content to 38.25 % even the culture medium contained elements. This results shown that sufficient carbon source and suitable nitrogen limitation facilitated PHB accumulation and prevented PHB degradation.

目錄
誌 謝 I
中文摘要 III
英文摘要 VI
目錄 VIII
表目錄 XII
圖目錄 XIV
第一章 前言 1
1-1 簡介 1
1-2 研究動機 2
1-3 論文架構 3
1-4 文獻回顧 4
1-4-1 生物可分解性塑膠 4
1-4-2 PHAs歷史簡介 5
1-4-3 PHAs之化學結構 7
1-4-4 PHAs之分解 8
1-4-5 PHAs之代謝途徑 9
1-4-6 PHAs之物理特性 10
第二章 實驗材料與方法 12
2-1 實驗材料 12
2-1-1 實驗藥品 12
2-1-2 實驗儀器 14
2-2 實驗方法 15
2-2-1 實驗菌株 15
2-2-3 培養基組成 15
2-2-4 搖瓶發酵實驗 17
2-3 培養基之探討 17
2-3-1 碳源之探討 17
2-3-2 複合氮源之探討 17
2-3-3 NaCl濃度之探討 18
2-3-4 添加其它離子之探討 18
2-4發酵槽之操作 18
2-5 菌體分析 20
2-5-1 菌體濃度之測定 20
2-5-2菌體乾重(CDW)之測定 20
2-6 PHA之分析 20
2-7 Glycerol殘餘量之分析 21
第三章 結果與討論 23
3-1 碳源基質之確立 23
3-1-1 碳源濃度之探討 23
3-2 複合氮源之探討 27
3-2-1 Yeast extract濃度之探討 27
3-2-2 Trypton濃度之探討 28
3-3 NaCl濃度之探討 31
3-4 添加離子之探討 33
3-5 不同pH值對PHB累積之影響 35
3-5-1 MGYT培養基以不同pH值之探討 35
3-5-2 MGYTT培養基以不同pH值之探討 35
3-6 調控pH值對PHB累積之探討 36
3-6-1 以MGYT medium進行pH値調控之探討 36
3-6-2以MGYTT medium進行pH値調控之探討 37
3-7 pH値對intracellular PHB depolymerase活性之影響 37
3-8 離子對PHB累積之影響 46
3-8-1 去除單一離子之探討 46
3-8-2 單一添加離子之探討 46
3-9 Fermentation strategy 50
3-9-1 Batch之探討 50
3-9-2 Fed-batch之探討 51
3-9-3 尋找最佳碳、氮比饋料液之探討 52
3-9-4 以不同pH值進行Fed-batch發酵之探討 53
3-9-5 Two stage之探討 54
3-10 Vibrio sp. M11於3L Fermenter培養之探討 65
3-10-1 以MGYT medium進行3L發酵槽培養之探討 65
3-10-2 以MGYTT medium進行3L發酵槽培養之探討 66
3-10-3 以3L Fermenter進行Fed-batch發酵策略之探討 66
3-10-4 調控pH値對3L Fermenter進行Fed-batch發酵策略之探討 67
第四章 結論與未來展望 72
4-1 結論 72
4-2 未來展望 73
參考文獻 74

Anderson A. J., Dawes E. A. Occurrence, metabolism, metabolic
role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiological Reviews., 54,450-472 (1990).

Byrom D. Polymer synthesis by microorganisms: technology and economics. Trends in biotechnology Referenceed., 5, 246-250(1987).

Bruce A., Juliana A., David G. Production of Poly-3-Hydroxyalkanoic Acid by Pseudomonas cepacia. Applied and Environmental Microbiology., 55, 584-589(1989).

Bertrand J.L., Ramsay B.A., Ramsay J.A., Chavarie C. Biosynthesis of Poly-beta-Hydroxyalkanoates from Pentoses by Pseudomonas pseudoflava. Applied and Environmental Microbiology., 56, 3133-3138(1990).

Chen G.Q., Wu Q., Zhao K., Yu P.H. Functional polyhydroxyalkanoates synthesized by microorganism. Chinese Journal of Polymer Science (English Edition)., 18, 389-396(2000).



Chen G.Q., Zhang G., Park S., Lee S.Y. Industrial scale production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Applied Microbiology and Biotechnology., 57, 50-55(2001).

Doi Y., Sudesh K., Abe H. Synthesis, structure and properties of polyhydroxyalkanoates:biological polyesters. Progress in Polymer Science (Oxford)., 25, 1503-1555 (2000).

Findlay R.H., White D.C. Polymeric beta-hydroxyalkanoates from environmental samples and Bacillus megaterium. Applied and Environmental Microbiology., 45, 71-78 (1983).

Horowitz D.M., Sanders J.K.M. Amorphous, Biomimetic Granules of Polyhydroxybutyrate:Preparation, Characterization, and Biological Implications. Journal of the American Chemical Society., 116, 2695-2702(1994).

Jendrossek D., Schirmer A., Schlegel H.G. Biodegradation of polyhydroxyalkanoic acids. Applied Microbiology and Biotechnology., 46, 451-463(1996).

Jung K., Hazenberg W., Prieto M., Witholt B. Two-Stage Continuous Process Development for the Production of Medium-Chain-Length Poly (3-Hydroxyalkanoates). Biotechnology and Bioengineering., 72, 19-24 (2001).

Jendrossek D., Handrick R., Reinhardt S., Kimmig P. The "intracellular" poly(3-hydroxybutyrate) (PHB) depolymerase of Rhodospirillum rubrum is a periplasm-located protein with specificity for native PHB and with structural similarity to extracellular PHB depolymerases. Journal of Bacteriology., 186, 7243–7253(2004).

Kim G.J., Lee I.Y., Yoon S.C., Shin Y.C., Park Y.H. Enhanced yield and a high production of medium-chain-length poly(3-hydroxyalkanoates) in a two-step fed-batch cultivation of Pseudomonas putida by combined useof glucose and octanoate. Enzyme and Microbial Technology., 20, 500-505(1997).

Lee S.Y. Bacterial Polyhydroxyalkanoates. Biotechnology and Bioengineering., 49, 1-14(1996).

Lee S.Y., Wang F. High Cell Density Culture of Metabolically Engineered Escherichia coli for the Production of Poly(3-hydroxybutyrate) in a Defined Medium. Biotechnology and Bioengineering., 58, 325-327(1997).

Lee S.Y., Choi J. High-Level Production of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) by Fed-Batch Culture of Recombinant Escherichia coli. Applied and Environmental Microbiology., 65, 4363–4368(1999).

Liu S.J. Biosynthesis and accumulation of poly(3-hydroxybutyrate) in Vibrio natriegens. Chinese journal of biotechnology., 18, 614-618(2002).

Ojumu T.V., Yu J. and Solomon B.O. Production of Polyhydroxyalkanoates, a bacterial biodegradable polymer. African Journal of Biotechnology., 3, 18-24(2004).

Poirier Y., Nawrath C., Somerville C. Production of polyhydroxyalkanoates, a family of biodegradable platics and elastomers, in bacteria and plants. Biotechnology., 13, 142-150 (1995).

Saito T., Saegusa H., Miyata Y., Fukui T. Intracellular degradation of poly(3-hydroxybutyrate) granules of Zoogloea ramigera I-16-M. FEMS Microbiology Letters., 103, 333-338(1992).

Sun W., Cao J.G., Teng K., Meighen E.A. Biosynthesis of Poly-3-hydroxybutyrate in the Luminescent Bacterium, Vibrio harveyi,a nd Regulation by the lux Autoinducer, N-(3-Hydroxybutanoyl)homoserineL actone. Journal of Biological Chemistry., 269, 20785-20790(1994).

Saito T., Takizawa K., Saegusa H. Intracellular poly(3-hydroxybutyrate) depolymerase in Alcaligenes eutrophus. Canadian journal of microbiology., 41, 187-191(1995).
Sun W., Teng K., Meighen E. Detection of poly(3-hydroxybutyrate) granules by electron microscopy of Vibrio harveyi stained with malachite green. Canadian journal of microbiology., 41, 131-137(1995).

Tsuge T. Metabolic Improvements and Use of Inexpensive Carbon Sources in Microbial Production of Polyhydroxyalkanoates. Journal of Bioscience and Bioengineering., 94, 579-584(2002).

Witholt B., De Smet MJ., Eggink G., Kingma J., Wynberg H. Characterization of intracellular inclusions formed by
Pseudomonas oleovorans during growth on octane. Journal of Bacteriology., 154, 870-878(1983).

Williamson D., Mellanby J., Krebs H. Enzymic Determination of D(- )- β-Hydroxybutyric Acid and Acetoacetic Acid in Blood. Biochemical Journal., 82, 90-96(1962).

黃晉貴. 台灣本土根瘤菌Wautersia taiwanensis 184生產聚羥 基丁酸發酵策略之探討, 生物科技暨生物資訊研究所碩士論文, 桃園(2005).

方智穎. 一系列聚-3-羥基烷酯之發酵生產及其發酵策略探討, 生物科技暨生物資訊研究所碩士論文, 桃園(2006).

周孟輝. 海洋環境中生產聚羥基烷酸微生物之探討, 生物科技暨生物資訊研究所碩士論文, 桃園(2006).