臺灣博碩士論文加值系統

己二烯二酸(Muconic acid,MA) 可於50 psi 及室溫下以鉑當觸媒轉換成己二酸(adipic acid)。己二酸為製造nylon 6,6等塑料之重要原料，目前以化學合成法為主，而過程中排放大量NOX等溫室氣體，會造成溫室效應。若以白色生物技術生產己二烯二酸，替代原本化學合成法，能降低能源消耗與環境污染。本實驗室由工業廢水中分離所得菌株，鑑定為Pseudomonas sp.，經突變得一高己二烯二酸產率菌株。
本研究由調整基質濃度、改良製程，探討提升產物累積及菌體增殖的效率。實驗發現當己二烯二酸濃度高於 10 g/L，便導致菌體增殖速率降低40%及降低生產力50%。調控最高苯甲酸濃度於 1.5g/L，能將比生長速率提升380%，並使平均生產力提高140%。基質濃度方面，當葡萄糖10 g/L，碳氮比為0.5時，比生長速率能達0.63 hr-1及最大單位菌體生產力0.86 g MA /g DCW/hr 。藉由提高轉換苯甲酸生成己二烯二酸期間溶氧值於30%以上，使最大單位菌體生產力提升約40% 。最後證實，基質中的苯甲酸會誘導Pseudomonas sp.中調控己二烯二酸代謝路徑基因之活性，於饋料批式程序中最終己二烯二酸累積濃度由 12 g/ L 提升至 19 g/L，並使生產力提高30 %，有助於饋料批式醱酵製程中己二烯二酸的累積。

Muconic acid (MA) can be transformed to adipic acid by using the Pt as catalytic agent at 50 psi and room temperature. Adipic acid is an important raw material for producing plastics such as nylon 6,6 which is produced through chemical synthesis; however, that chemical manufacturing will cause a lot of greenhouse gases of NOx to be discharged during the process thus adding to the greenhouse effect. Energy consumption and environmental pollution can be reduced if white biotechnology is used to replace the chemical synthesis to produce the MA. Our laboratory obtained a bacterial strain from the sludge of industrial wastewater treament. The strain, identified to be Pseudomonas sp., has been mutated to a high-yield MA strain by chemical mutagens and proper screening strategy. We found the compositions of the complete medium by using the response surface methodology, further discussed the impacts of the mass transfer of oxygen in fermentation batch on the generation of MA, and the comparison of the compound nutrition assembly used in fed-batch cultivation.
The goal of this study is to discuss how to improve the efficiency of product accumulation and cell growth after amplifying the MA procedure by adjusting the concentrations of substrate and improving the procedures. We studied the inhibition level of the product concentration on the MA produced by cells, the test found that the speed of cell growth was reduced by 40% and the productivity was decreased by 50% when the MA concentration was higher than 10g/L; and the cellular productivity could be improved to 1.2 g MA /g DCW/hr by keeping the dissolved oxygen value above 30% during transference of the benzoic acid to generate MA. In the aspect of procedural improvement, variable feeding is chosen to replace periodic feeding. It can not only improve the specific growth rate by 380%, but also increase the original productivity by 140%. In the aspect of substrate concentrations, the proper glucose concentration and C/N ratio are used to achieve the aims of cell growth and productivity improvement: When the glucose concentration is 10 g/L with 0.5 of C/N ratio, the specific growth rate can reach 0.63 hr-1 and the cellular productivity is 0.86 g MA /g DCW/hr.
Finally, this process verifies that the addition of benzoic acid in the batch stage can induce the activity of the genes used to regulate and control the metabolism pathway of MA production in Pseudomonas sp., which is conducive to the accumulation of MA in the fed-batch fermentation.

目錄
中文摘要...................................................................................................................i
Abstract.....................................................................................................................ii
誌謝.........................................................................................................................iv
目錄.........................................................................................................................v
圖目錄...................................................................................................................viii
表目錄....................................................................................................................xi
第一章 緒論.............................................................................................................1
1-1前言...................................................................................................................1
1-2苯甲酸與葡萄糖的生物降解路徑…………………………………………..2
1-2-1 苯甲酸生物降解生成己二烯二酸……………………………………2
1-2-2 葡萄糖生物轉換生成己二烯二酸……………………………………4
1-3 琥珀酸於Pseudomonas sp.生成己二烯二酸扮演之角色…………………6
1-4 研究動機與目的…………………………………………………………….8
第二章 文獻回顧……………………………………… ……………………….10
2-1 微生物醱酵生產己二烯二酸………………………………………….......10
2-2 基因工程應用於己二烯二酸的生產…………………………………...…12
第三章 實驗材料與方法………………………………………………………...15
3-1 實驗材料…………………………………………………………………...15
3-1-1 實驗藥品……………………………………………………………..15
3-1-2 己二烯二酸粗製備…………………………………………………..15
3-2 儀器與實驗設備…………………………………………………………...16
3-3 實驗菌株與培養基……………………………………………...…………17
3-3-1菌株來源……………………………………………………………...17
3-3-2固態培養基…………………………………………………….…..…18
3-3-3 液態培養基……………………………………………………….…18
3-4 菌種活化與培養……………………………………………………….…..19
3-4-1菌種活化……………………………………………………...………19
3-4-2前陪養……………………………………………………………...…19
3-4-3醱酵槽前置作業…………………………………………………...…19
3-5 分析方法…………………………………………………………………...19
3-5-1菌量測定……………………………………………………………...19
3-5-2己二烯二酸、苯甲酸、鄰苯二酚測定………………………………20
3-5-3琥珀酸測定………………………………………………………...…23
3-5-4葡萄糖測定…………………………………………………………...24
3-5-5氮源測定……………………………………………………………...25
3-6 醱酵實驗之重要參數……………………………………………………...26
第四章 結果與討論……………………………………………………………...29
4-1 產物回饋抑制……………………………………………………………...29
4-1-1實驗設計……………………………………………………………...30
4-1-2己二烯二酸濃度對菌體成長及產物生成之抑制程度……………...31
4-2 饋料期間苯甲酸濃度對醱酵生產己二烯二酸之影響………………….. 36
4-3 碳氮比對Pseudomonas sp.醱酵生產己二烯二酸之影響………………...38
4-3-1碳氮比對菌體增殖、生產力及單位菌體生產力之影響……………39
4-3-2葡萄糖濃度對己二烯二酸產量之影響……………………………...41
4-4 溶氧值對醱酵生產己二烯二酸之影響…………………………………...45
4-5 批式培養中苯甲酸濃度對己二烯二酸生成之影響……………………...47
第五章 結論……………………………………………………………………...50
第六章 未來展望………………………………………………………………...52
參考文獻………………………………………………………………………….53
附錄一…………………………………………………………………………….56
附錄二…………………………………………………………………………….57
附錄三…………………………………………………………………………….58
附錄四…………………………………………………………………………….74
附錄五…………………………………………………………………………….78
圖目錄
圖 1-1 化學合成法示意圖……………………………………………………...2
圖 1-2 苯甲酸代謝路徑圖……………………………………………………...3
圖 1-3 葡萄糖轉換己二烯二酸之生物降解路徑圖…………………………...5
圖 1-4 三羧酸循環示意圖……………………………………………………...7
圖 2-1 Conversion of benzoate to cis, cis-muconate by P. putida KT2440-JD1..
…………………………………………………………………………12
圖 3-1 自行製備己二烯二酸………………………………………………….16
圖 3-2 光學密度(OD) 與菌體量(DCW)關係圖……………………………...20
圖 3-3 己二烯二酸(MA)、苯甲酸(BA)與鄰苯二酚(CA) HPLC分析之圖譜…..
…………………………………………………………………………21
圖 3-4 己二烯二酸檢量線…………………………………………………….21
圖 3-5 苯甲酸檢量線………………………………………………………….22
圖 3-6 鄰苯二酚檢量線……………………………………………………….22
圖 3-7 琥珀酸(SA)之HPLC分析圖譜……………………………………….23
圖 3-8 琥珀酸檢量線………………………………………………………….24
圖 3-9 葡萄糖(G)檢量線………………………………………………………25
圖 3-10 菌量增殖(a)、產物生成(b)、基質消耗(c)示意圖……………………..28
圖 4-1 饋料批式醱酵之菌體、基質、產物歷時圖………………………….30
圖 4-2 批式醱酵之菌體、基質、產物歷時圖………………………………..31
圖 4-3 己二烯二酸濃度與比生長速率關係圖……………………………….32
圖 4-4 己二烯二酸濃度與最終總菌量之關係圖…………………………….32
圖 4-5 己二烯二酸濃度與己二烯二酸生成速率關係圖…………………….33
圖 4-6 己二烯二酸濃度與苯甲酸消耗速率關係圖………………………….34
圖 4-7 己二烯二酸濃度與單位菌體轉化率之關係圖…………………….35
圖 4-8 己二烯二酸濃度與基質轉化率之關係圖…………………………...35
圖 4-9 饋料期間調控苯甲酸之歷時圖……………………………………...37
圖 4-10 饋料期間調控不同苯甲酸濃度之菌量增殖歷時圖………………...37
圖 4-11 饋料期間調控不同苯甲酸濃度之己二烯二酸歷時圖……………...38
圖 4-12 葡萄糖濃度與不同碳氮比其菌體量與生產力之關係圖…….…….42
圖 4-13 不同葡萄糖濃度之碳氮比與單位菌體生產力之關係圖…………43
圖 4-14 不同葡萄糖濃度之碳氮比與比生長速率之關係圖………………..43
附錄 1 產物回饋抑制探討，各組實驗菌體量、己二烯二酸、苯甲酸歷時圖...56
附錄2-1 饋料批式實驗調控最高苯甲酸濃度於5g/L之實驗歷時圖…...…….57
附錄2-2 饋料批式實驗調控最高苯甲酸濃度於1.5g/L之實驗歷時圖…….....57
附錄3-1 碳氮比4、葡萄糖5 g/L之醱酵歷時圖…….…………………...……...58
附錄3-2 碳氮比2、葡萄糖5 g/L之醱酵歷時圖…..……………………..……...59
附錄3-3 碳氮比1、葡萄糖5 g/L之醱酵歷時圖………………………....……...60
附錄3-4 碳氮比0.5、葡萄糖5 g/L之醱酵歷時圖………………………….......61
附錄3-5 碳氮比0.25、葡萄糖5 g/L之醱酵歷時圖…………………....….…….62
附錄3-6 碳氮比4、葡萄糖10 g/L之醱酵歷時圖………………………..……...63
附錄3-7 碳氮比2、葡萄糖10 g/L之醱酵歷時圖………………………..……...64
附錄3-8 碳氮比1、葡萄糖10 g/L之醱酵歷時圖………………………..….......65
附錄3-9 碳氮比0.5、葡萄糖10 g/L之醱酵歷時圖……………………...……...66
附錄3-10 碳氮比0.25、葡萄糖10 g/L之醱酵歷時圖…………………….......67
附錄3-11 碳氮比4、葡萄糖20 g/L之醱酵歷時圖…………….……..……...68
附錄3-12碳氮比2、葡萄糖20 g/L之醱酵歷時圖…………………..........…69
附錄3-13 碳氮比1、葡萄糖20 g/L之醱酵歷時圖…………………...…...…70
附錄3-14 碳氮比0.5、葡萄糖20 g/L之醱酵歷時圖……………………...….71
附錄3-15 碳氮比0.25、葡萄糖20 g/L之醱酵歷時圖……………………..….72
附錄3-16 對照組(葡萄糖 1.5 g/L、酵母萃取粉4.9 g/L) 之醱酵歷時圖..….....73
附錄4-1 葡萄糖 20g/L、酵母萃取粉 5g/L、溶氧調控10%-20%以上之醱酵歷時圖…………………………………………………………………….74
附錄4-2葡萄糖10g/L、酵母萃取粉5g/L、溶氧調控10%-20%之醱酵歷時圖………………………………………………………………….……75
附錄4-3葡萄糖20g/L、酵母萃取粉 5g/L、溶氧調控30%以上之醱酵歷時圖……….………………………………………………………………76
附錄4-4葡萄糖 10g/L、酵母萃取粉 5g/L、溶氧調控30%以上之醱酵歷時圖……..……………………………………………………………...…77
附錄5-1 醱酵初始無添加苯甲酸之菌體、產物、基質、溶氧及轉速歷時圖….78
附錄5-2 醱酵初始添加苯甲酸之菌體、產物、基質、溶氧及轉速歷時圖……...79
表目錄
表 2-1 各研究團隊與本實驗室目前醱酵生產己二烯二酸之統整表……….14
表 3-1 實驗藥品一覽………………………………………………………….15
表 3-2 實驗設備一覽………………………………………………………….16
表 3-3 固態培養基成分(以NaOH調至pH7)………………………………..18
表 3-4 培養基成分(以NaOH調至pH7)……………………………………..18
表 4-1 葡萄糖濃度與不同碳氮比之比生長速率、生產力、單位菌體轉化率…
……………………………………………………………………….…44
表 4-2 葡萄糖濃度與己二烯二酸生成量之關係…………………………….45
表 4-3 溶氧與菌體量、單位菌體轉化率之關係…………………………….47
表 4-4 醱酵初始有無添加苯甲酸於生成己二烯二酸各項參數之比較…….49

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