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研究生:辛文富
研究生(外文):Wen-Fu Xin
論文名稱:玉米漿應用於本土菌株PseudomonasaeruginosaJ16醱酵生產鼠李醣脂之探討
論文名稱(外文):Use of corn steep liquor on production of rhamnolipid with an indigenous isolate Pseudomonas aeruginosa J16
指導教授:魏毓宏
學位類別:碩士
校院名稱:元智大學
系所名稱:生物科技與工程研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:122
中文關鍵詞:鼠李醣脂綠膿桿菌回應曲面法饋料批次玉米漿
外文關鍵詞:RhamnolipidP. aeruginosaRSMCorn steep liquor
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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%。
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.
目錄
中文摘要 Ι
英文摘要 IIΙ
致謝…………………………………………………………………...…V
目錄………………………………………………………………..……VI
表目錄……...…………………………………………………………....X
圖目錄……………………………………………………………….…XII
第一章 文獻回顧 1
1-1生物界面活性劑 1
1-2微生物生產生物界面活性劑之篩選 3
1-3生物界面活性劑之種類 4
1-3-1醣脂類…………...…..………………………………….....5
1-3-2磷脂類與脂肪酸……………………...………...................6
1-3-3脂胜肽………………………………………………….….7
1-3-4高分子生物界面活性劑…………………………………..7
1-3-5微粒型生物界面活性劑………………………………..... 8
1-4生物界面活性劑之應用…………………………………....…...14
1-5生物界面活性劑之純化回收…………………..……………….18
1-6鼠李醣脂之基因合成與代謝路徑之探討……………..……….20
1-7 鼠李糖脂之應用與發展……………….……………………….24
1-8玉米漿基質組成… 25
1-8-1玉米漿基質之製備 26
1-8-2玉米漿基質之應用 26
1-9實驗設計-回應曲面法 32
1-9-1 二水準因數設計……………………...…...…………….33
1-9-2 陡升路徑法 34
1-9-3 中心混成設計 34
1-9-4 回應曲面模式適切性之統計試驗 35
1-9-5 檢定係數R2 35
1-10研究動機及目的………………..…………………………..….39
第二章、材料與方法 41
2-1 實驗藥品 41
2-2 實驗儀器……………………………………………………….43
2-3菌株及培養基 44
2-3-1 實驗菌株…………………………………………..…….44
2-3-2 培養方法………………………………………….……..44
2-3-3 培養基組成……………………………………………...44
2-4 鼠李醣脂之分離純化…………………………………...……...47
2-5鼠李醣脂(Di-rhamnolipid)之分離…………………………...….48
2-6實驗步驟與分析方法…………………………...……………….50
2-6-1鼠李醣脂之定性分析………………………………...….50
2-6-2 鼠李醣脂之定量分析………………………...…………50
2-6-3 甘油定量分析 51
2-6-4 菌體生長分析 52
2-6-5 酸鹼值測定 52
第三章、結果與討論 54
3-1 論文架構 54
3-2 培養基最適化 55
3-2-1複合式營養源額外添加於無機鹽培養基……………....55
3-2-2 複合式營養源取代無機鹽培養基中無機氮源 56
3-2-3複合式營養源取代無機鹽培養基中碳源 56
3-2-4 以實驗設計進行培養基組成之最適化探討…………...60
3-2-5 最適化半合成培養基與最適化無機鹽培養基之比較………………………………………………………...71
3-3 屬李醣脂之定性分析…………………………………………..75
3-4玉米漿與鼠李醣脂之探討……………………………………...82
3-5 發酵策略…………………………………………………..…...88
3-5-1 批次醱酵………………………………………...……....88
3-5-2 饋料批次醱酵…………………………………………...90
3-5-3饋料方式之探討………………………………...…..….100
3-6鼠李醣脂(Di-rhamnolipid)之分離……………………………..108
第四章 結論與展望………………………..……………………...….112
4-1結論……………………………………………………….....…112
4-2展望…………………………………………………………….115
參考文獻………………………………………………………………116

表目錄
表1-1 微生物生產界面活性劑的種類及物理性質……………………9
表1-2常見生物界面活性劑的回收製成 19
表1-3 微生物在玉米漿生長情形…………………………………......29
表1-4玉米漿成份分析表………………………………………………29
表1-5玉米漿中灰份成份組成……………………………………........30
表1-6玉米漿之應用……………………………………………………31
表2-1 LB 培養基之成份組成................................................................45
表2-2無機鹽培養基之成份組成............................................................45
表2-3最適化無機鹽培養基之成份組成 46
表2-4最適化半合成培養基之成份組成................................................46
表3-1 Two-level 實驗設計表………………………………………….64
表3-2 Two-level 實驗設計以Plackeet-burman foled mehod 之實驗結果………………………………………………………….........66
表3-3 Two-level 實驗設計以Plackeet-burman foled mehod 回歸分析結果………………………………………………………….....66
表3-4 陡升路徑之實驗設計表………………………………………..67
表3-5 RSM實驗設計以Box-Behnken method之實驗結果………….68
表3-6 RSM實驗設計以Box-Behnken method 之回歸分析表……….68
表3-7 最適化半合成培養基與無機鹽培養基產率之比較…………..73
表3-8最適化無機鹽培養基與半合成培養基中Di-rhamnolipid 與 mono-rhamnolipid之比例……………………….……………..73
表3-9 鼠李醣脂碳譜主要化學位移…………………………….….....79
表3-10在相同時間下(24h)不同濃度的玉米漿對甘油使用率及鼠李醣脂產率………………………………………………….…....86
表3-11不同饋料方式之產率比較……………………….………..….106
表3-12 利用C-18來單獨取得Di-rhamnolipid之實驗結果………....110
表3-13 利用LC-Silica來單獨取得Di-rhamnolipid之實驗結果…....110

圖目錄
圖1-1 P. aeruginosa 生產鼠李醣脂之主要有六種異構物 10
圖1-2 利用Rhodococcus erythopolis發酵生產Trehalolipids 11
圖1-3 利用Torulopsis bombicola發酵生產Sorphorolipids 11
圖1-4 利用Bacillus subtilis 發酵生產Surfactin 12
圖1-5利用Acinetobact r sp.發酵生產phosphatidylethanolamine 12
圖1-6 利用Acinetobacter calcoaceticus RAG-1發酵生產heterpolysaccharide 13
圖1-7 生物界面活性劑移除重金屬之機制 17
圖1-8 鼠李醣脂之基因調控及合成 22
圖1-9 鼠李醣脂之代謝路徑 23
圖1-10 玉米漿的製備………..………………………...……………...28
圖1-11 回應曲面圖 36
圖1-12 反應曲面法設計提成圖 37
圖1-13 RSM設計之概念X1(1)~(8)為二水準因數之實點;(9)~(15)為中心混成設計法之補充實驗點 38
圖1-14 陡升路徑概念圖 38
圖2-2 分離Di-rhamnolipid之示意圖……………………………….....49
圖2-3 鼠李醣脂經HPLC所得之校正曲線………………..……….....53
圖2-4 甘油經HPLC所得之校正曲線……………………...…..……..53
圖2-5 菌體光學密度與細胞乾重之校正曲線……………...………...53
圖3-1 論文架構…………………………...…………………………...54
圖3-2 複合式營養源額外添加於無機鹽培養基………………..........57
圗3-3 複合式營養源取代無機鹽培養基中無機氮源..………………58
圖3-4 複合式營養源取代無機鹽培養基中碳源..……………………59
圗3-5 篩選甘油濃度-1及+1……………………...…..……………….65
圗3-6 篩選硫酸胺濃度-1及+1……………………….....…………….65
圗3-7篩選玉米漿濃度-1及+1………………………………...……….65
圖3-8 陡升路徑圖..……………………………………………………67
圖3-9 甘油和硫酸胺對鼠李醣脂產量之曲面圖……………..………69
圖3-10 甘油和玉米漿對鼠李醣脂產量之曲面圖…………………....69
圖3-11硫酸胺和玉米漿對鼠李醣脂產量之曲面圖…………….….…70
圖3-12 最適化培養基之三重複實驗……………………...………….70
圖3-13 最適化半合成培養基與無機鹽培養基對菌體生長、rhamnolipid合成與甘油使用率之比較………...…………....74
圖3-14 利用100%乙醇回溶所得的鼠李醣脂………………......……77
圖3-15 利用50%乙醇回溶所得的鼠李醣脂………………...……….77
圖3-16 鼠李醣脂以1H-NMR所得之圖譜……………………….....…78
圖3-17 鼠李醣脂以13C-NMR所得之圖譜 ………………………....78
圖3-18 利用P. aeruginosa J16 經醱酵及分離純化所得之鼠李醣脂FAB/MS分析圖譜…………………………............................80
圖3-19 利用P. aeruginosa J16 經醱酵及分離純化所得之鼠李醣脂經HPLC分析圖譜…………...……………………………….…81
圖3-20 玉米漿對鼠李醣脂合成路徑之關係……………………......85
圖3-21 探討相同時間之下不同玉米漿對甘油使用率及鼠李醣脂產率………………………………………………..…………..87
圖3-22 P. aeruginosa J16 在最適化培養基中菌體生長、營養源的消耗、菌體容氧量與生產鼠李醣脂之關係………..….…………89
圖3-23 工業級與試藥級甘油對菌體與鼠李醣脂之影響……………94
圖3-24 饋料時間點對P. aeruginosa生長與鼠李醣脂產量之影響….95
圖3-25 饋料成份對P. aeruginosa生長與鼠李醣脂產量之影響….…96
圖3-26不同前驅物進行饋料對菌體生長與鼠李醣脂之影響………..97
圖3-27不同前驅物進行饋料對菌體生長與鼠李醣脂之影響………..98
圖3-28 添加不同濃度營養源對鼠李醣脂之影響………………...….99
圖3-29 以固定時間添加營養源之發酵策略………………………..103
圖3-30以菌體濃度變化添加營養源之發酵策略……………...…….104
圖3-31以pH值變化添加營養源之發酵策略……………...…..…….105
圖3-32 不同饋料方式對菌體生長與鼠李醣脂合成之比較…..…....107
圖3-33 利用C-18來單獨取得di-rhamnolipid經HPLC分析之圖譜.111
圖3-34 利用LC-Silica取得di-rhamnolipid經HPLC分析之圖譜…..111
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