利用海洋弧菌Vibrio sp.M11生產聚羥基丁酯之探討

Production of poly(3-hydroxybutyrate) by Vibrio sp. M11

黃宖堉(Hung-Yu Huang)

指導教授 ： 魏毓宏

元智大學/工程學院/生物科技暨生物資訊研究所/碩士(2007年)

摘要

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。

關鍵字

海洋弧菌 ； 聚羥基烷酯 ； 聚羥基丁酯 ； 發酵策略

並列摘要

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.

並列關鍵字

Vibrio sp. M11 ； Polyhydroxyalkanoates ； Polyhydroxybutyrate ； Fermentation strategy

參考文獻

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