玉米漿應用於本土菌株Pseudomonas aeruginosa J16醱酵生產鼠李醣脂之探討

Use of corn steep liquor on production of rhamnolipid with an indigenous isolate Pseudomonas aeruginosa J16

辛文富(Wen-Fu Xin)

指導教授 ： 魏毓宏

元智大學/工程學院/生物科技與工程研究所/碩士(2008年)

摘要

本研究主要是利用石化廢水篩選獲得之台灣本土菌株Pseudomonas aeruginosa J16，探討其代謝生產鼠李醣脂(rhamnolipid)之相關培養基。研究發現：培養基中添加玉米漿(con steep liquor, CSL)，將有助於菌體生長及誘發產物鼠李醣脂之代謝。研究進一步顯示：0.44 % CSL添加於無機鹽培養基，促進鼠李醣脂濃度達7.68 g/L，相較於未添加CSL控制組分別提升約1.87倍。本研究進一步利用回應曲面實驗設計法(response surface methodology, RSM)，求得最佳化生產鼠李醣脂培養基組成為：Glycerol (11.37 %)、(NH4)2SO4 (89.82 mM)、Corn steep liquor (3.74%)、NaHPO4 (40 mM)、KH2PO4 (40 mM)、CaCl2．2H2O (6 μM)、MgSO4．7H2O (577 μM)、EDTA (4 μM)、FeSO4．7H2O (4 mΜ)。P. aeruginosa J16利用上述回應曲面實驗設計所得之最適化培養基，能將鼠李醣脂產量提升至20.25 g/L，且最高產率可達0.210 g/L/h。所得的高產量產物經由核磁共振光譜儀、高效能液相層析儀及快速原子撞擊質譜儀分析即為鼠李醣脂。
在饋料批次策略上，以36小時進行饋料3%營養源，其營養源成份包含corn steep liquor 、glycerol 及 (NH4)2SO4，鼠李醣脂產量可達最好，產量為26.80 g/L。而在不同饋料策略方式中，以pH值變化進行饋料作為添加營養源時機，成功的得到鼠李醣脂最佳產量31.39 g/L， 為批次實驗的1.5倍，為無機鹽培養基之7.85倍。此結果顯示，以以pH值變化進行饋料作為添加營養源時機，為一方便且具效率之醱酵方式。
在鼠李醣脂分離實驗中，利用column中填充C-18進行mono-rhamnolipid與di-rhamnolipid之分離，成功的得到單一di-rhamnolipid，其5g的rhamnolipid進行分離，可收集到單一di-rhamnolipid 0.988 g，其回收率為19.76％。

關鍵字

鼠李醣脂 ； 綠膿桿菌 ； 回應曲面法 ； 饋料批次 ； 玉米漿

並列摘要

Pseudomonas aeruginosa J16 isolated from wastewater of the petrochemical factory located in southern Taiwan was used in this experimental study for the production of rhamnolipid. Recently researchers reported that, addition of 0.44%corn steep liquor(CSL) to the mineral salt medium enhanced the rhamnolipid production to 7.68 g/L, approximately 1.87-fold times higher than culture medium with no corn steep liquor.The optimal formulation was identified by response surface methodology (RSM) as follows: glycerol(11.37%), (NH4)2SO4(89.82mM), Corn steep liquor(3.74%), Na2HPO4 (40 mM), KH2PO4 (40 mM), CaCl2 (6 ?嵱), MgSO4.7H2O (577 ?嵱), EDTA (4?嵱), and FeSO4.7H2 (4?嵱). Statistical experimental design methodology was not only applied to optimize the culture medium composition also it was applied for further enhancement of rhamnolipid. Interestingly while applying this methodology the rhamnolipid concentration was 20.25 g/L and productivity of about 0.210 g/L/h have been achieved. The result of NMR、FAB-MS and HPLC analysis concealed that the biosurfactant is rhamnolipid
The results of fed-batch in shaking flask revealed that when optimum fed nutrient is 3% glycerol ((NH4)2SO4, corn steep liquor included) and the optimum fed timing is 36hr, the rhamnolipid production was 26.80 g/L . Significantly, feeding strategy result shows the best rhamnolipid concentration of 31.39 g/L, approximately 1.5-fold times higher than batch experiment and 7.85 fold times higher than mineral salt medium. These results once again confirmed that the fed batch strategy was convenient and efficient method for rhamnolipid production.
For efficient separation of mono-rhamnolipid and di-rhamnolipid a column with C-18 packing have been carried out. The results of separation revealed that 5g rhamnolipid may collect sole di-rhamnolipid 0.988 g and the recovery yield is 19.76% efficiently.

並列關鍵字

Rhamnolipid ； P. aeruginosa ； RSM ； Corn steep liquor

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