一系列聚-3-羥基烷酯之醱酵生產及其醱酵策略探討

Fermentative strategies for microbial production of a series of poly-(3-hydroxyalkanoates)

方智穎(Chih-Ying Fang)

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(2006年)

摘要

本研究主要利用Cupriavidus taiwanensis、Burkholderia sp.一系列本土根瘤菌、重組 E. coli以及Pseudomonas putida KT2442來生產一系列生物可分解塑膠聚羥基烷酯 (Polyhydroxyalkanoate, PHA)，其中包含短碳鏈的聚羥基烷酯(scl-PHA)以及中長碳鏈的聚羥基烷酯(mcl-PHA)。
首先利用Sudan Black B染色以此快速鑑定是否具有潛力生產PHA，其次使用氣相層析法(Gas chromatography, GC)分析鑑定PHA的種類，經過上述步驟挑選出Cupriavidus taiwanensis 184和Burkholderia sp. PTU9較具生產Poly-3-hydroxybutyrate (P(3HB))的潛力。分別進行其培養基之最適化、生長條件之最適化以及各種不同之醱酵策略，由搖瓶的測試後再經醱酵槽策略的應用使PHA產量做進一步的提升。
當第二階段培養溫度改變或額外添加重金屬，確實會誘發菌體累積合成P(3HB)。研究進一步顯示：當第二階段生長溫度由30℃上升至42℃，菌體合成P(3HB)含量將由原來37.2%提升至42.5%。其次，在重金屬添加方面，額外添加50μM K2Cr2O7及100μM CdCl2， P(3HB)含量分別可提升7.1％、9.4％，達41.3%及43.6%。
Burkholderia sp. PTU9在限氮批次醱酵下可得到菌體乾重達13.51 g/L ，其 P(3HB)含量達53.79 %，P(3HB)濃度可達7.26 g/L。重組 E. coli 在饋料批次醱酵策略下可得到菌體乾重達80.8 g/L，P(3HB)含量達40.6%，P(3HB)濃度為32.8 g/L。Pseudomonas putida KT2442在饋料批次醱酵策略下可得到菌體乾重可達36.36 g/L，PHA含量最高可達59.3%，其中包含Polyhydroxyhexanoate (PHHx) 3.38%、Polyhydroxyoctanoate (PHO) 24.13%、Polyhydroxydecanoate (PHD) 22.42%、Polyhydroxydodecanoate (PHDD) 9.43%。

關鍵字

Cupriavidus taiwanensis ； Burkholderia sp. ； 聚羥基烷酯 ； PHA ； Sudan Black B染色 ； Poly-3-hydroxybutyrate ； Polyhydroxyhexanoate ； Poly-hydroxyoctanoate ； Polyhydroxydecanoate ； Poly-hydroxydodecanoate ； 饋料批次醱酵策略

並列摘要

In this study we used Cupriavidus taiwanensis, Burkholderia sp. a series of nodule bacteria of native country, recombined E. coli and Pseudomonas putida KT2442 to produce a series of biodegradable polymer, Polyhydroxyalkanoate (PHA), included short chain length PHA (scl-PHA) and medium chain length (mcl-PHA). By Sudan Black B stain technique to determine which strain had a potential to produced PHA and used Gas chromatography (GC) to analyzed kinds of PHA. Cupriavidus taiwanensis 184 and Burkholderia sp. PTU9 were selected had a relatively potential to produced Poly-3-hydroxybutyrate (P(3HB)). The end point of this experiment was the optimization of culture medium, growth condition and all kinds of fermentative strategies. From flask test to the application of fermentor strategies, thus the productivity of PHA would be increased. At the second stage, switched of the temperature or added additional heavy metal, actually would induce accumulation of P (3HB) in bacteria.
The further study showed when growth temperature increased from 30℃ to 42℃ at the second stage, P(3HB) content would be improved from originally 37.2% to 42.5%. Secondly, added additional heavy metal, with 50μM K2Cr2O7 and 100μM CdCl2, P(3HB) content was differentiated and can be improved by 7.1%, 9.4%, enhanced to 41.3% and 43.6%, respectively. Burkholderia sp. PTU9 could get the cell dry weight enhanced to 13.51 g/L under the circumstance of the batch with limited nitrogen, its P(3HB) content reached 53.79%, P(3HB) concentration can reached 7.26 g/L. When used recombined E. coli in fed-batch fermentation strategy, could get cell dry weight up to 80.8 g/L, P(3HB) content up to 40.6%, P(3HB) concentration up to 32.8 g/L. While when use Pseudomonas putida KT2442 in fed-batch fermentation strategy, could get cell dry weight up to 36.36 g/L, PHA content can reached as high as 59.3%, included Polyhydroxyhexanoate (PHHx) 3.38%, Polyhydroxyoctanoate (PHO) 24.13%, Polyhydroxydecanoate (PHD) 22.42% and Polyhydroxydodecanoate (PHDD) 9.43%.

並列關鍵字

Cupriavidus taiwanensis ； Burkholderia sp. ； Polyhydroxy- hexanoate ； PHA ； Sudan Black B staining ； Poly-3-hydroxy- butyrate ； Polyhydroxyhexanoate ； Polyhydroxyoctanoate ； Polyhydroxydecanoate ； Polyhydroxydodecanoate ； Fed-batch Fermentative strategy

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