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研究生:巴 拉 莫
研究生(外文):Pramod Shah
論文名稱:重組大腸桿菌在有氧環境下利用粗甘油生產聚羥基烷酯與生物酒精
論文名稱(外文):Development of recombinant Escherichia coli for bioethanol/ poly-3-hydroxybutyrate production from crude glycerol under aerobic condition
指導教授:藍祺偉
指導教授(外文):John Chi-Wei Lan
口試委員:劉嚞睿林昀輝魏毓宏
口試委員(外文):Je-Ruei LiuYun-Huin LinYu-Hong Wei
口試日期:2013-07-15
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:2013
畢業學年度:101
語文別:英文
論文頁數:100
中文關鍵詞:大腸桿菌耗氧發酵粗甘油生物乙醇聚3-羥基丁酯醛還原酶乙醛脫氫酶
外文關鍵詞:Escherichia coliaerobic fermentationcrude glycerolbioethanolpoly 3-hydroxybutyratealdehyde reductasealdehyde dehydrogenase
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由於能源危機、化石燃料價格的波動及環保意識的提升,導致開發替代燃料及再生能源成為一個永續發展課題。而生質柴油為一種被開發來做為替代燃料的能源,粗甘油則為生產生質柴油時產生的副產物,近幾年來大量的開發生質柴油也造成粗甘油的價值低於市場價值。粗甘油對市場經濟的影響已經成為生質柴油對經濟的危害,因而近代開始研究如何使用粗甘油來增進其市場價值。近年來以甘油作為發酵過程中的原料被大量的關注,但粗甘油因雜質存在下,會對生物轉換產生負效益影響。因此在過去的一些研究中以純甘油作為發酵原料,但使用粗甘油還是可以降低營運成本。大腸桿菌為一種常用於生物產業的菌種,且已經利用於將粗甘油轉換成有價值的產品。重組大腸桿菌可在有氧條件下把粗甘油轉換生產乙醇跟聚3-羥基丁酯。
本研究分為兩部分,第一部分是探討醛還原酶(ALRD)與乙醛脫氫酶(ALDH)的代謝途徑。在這一部分中,發現重組大腸桿菌BL-21_pARD33對粗甘油的利用幾乎高出一般野生型大腸桿菌兩倍。結果表明在有氧的條件下,並無較多的生物量下,且甘油的利用量有顯著的增加。此外,這兩種酶產生了新的氧化還原替代途徑產生生物乙醇且醋酸作為中間產物累積。
醋酸的毒性和氧氣的限制可能會造成細胞生長的阻礙,故以此作為我的研究基礎,接續於第二部分中。我轉殖的第二質體含有VGB基因與phaCABRE 操縱因子於大腸桿菌BL-21_pARD33中。在批次培養中,高溶氧的條件下解決了氧氣擴散的限制,可藉由VGB的基因使得生物有更高的生物量。接著醋酸將由phaCAB操縱因子的第二個途徑將acetyl-CoA轉化成P3HB。在這一部分中,我們發現重組大腸桿菌BL-21_pARD33-BHB2因含有這兩個質體能更有效率的利用粗甘油來生產生物乙醇跟聚3-羥基丁酯。儘管這兩個產物競爭使用相同的碳源,但此競爭為有益的競爭,因為他的甘油利用量較一般型大腸桿菌高出了兩倍,並能提高產物的產量。而此重組大腸桿菌中最大的生產為17.4mmol L-1 的生物乙醇與30.2%的乾菌重的聚3-羥基丁酯。
最後,我調查這兩個質體的穩定性,在不同條件下(未經誘導的LB培養基與Define培養基),結果顯示,在培養96小時後,大腸桿菌BL21_pARD33-BHB2仍有超過50%的穩定性。
Environmental awareness, energy crisis along with fluctuation in price of fossil fuels, a driving necessity is prompted for the search and development of alternative fuel from renewable resources that are sustainable, regenerative and ecologically friendly. The production of biodiesel, one of the promising of biomass fuels account in generation of inevitable waste product, crude glycerol. The mandatory demand of biodiesel in recent years has lead with huge flood of crude glycerol, fading its market value (price) worldwide. Crude glycerol has created burden in terms of disposal and economic viability in biodiesel industries. Hence, utilization of this abundant crude glycerol to several value added products is contemporary research area with beneficial features. Recently, the use of crude glycerol in fermentation process is one of the prominent feedstock gaining lots of attention. However, the presence of lower amount of glycerol with several impurities in crude glycerol has been expected to influence the negative effect in bioconversion process. Thus, several researches in past, have been performed with pure glycerol as a fermentation substrate but now the use of crude glycerol is focused for reducing the operational cost. Escherichia coli, a model organism in bioindustries, has been engineered by several researchers for the conversion of crude glycerol to value products. In this fashion, recombinant Escherichia coli strains were constructed for the simultaneous production of bioethanol and poly 3-hydroxybutyrate (P3HB) from crude glycerol, under aerobic condition.
My dissertation is divided into two parts where in the first part the metabolic pathways introduced by aldehyde reductase (Alrd) and aldehyde dehydrogenase (AldH) were investigated. In this part I found that the recombinant strain Escherichia coli BL21_pARD33 could nearly utilize 2-fold higher crude glycerol than that of wild-type {Escherichia coli BL21 (λDE3)}. The results showed significant utilization of crude glycerol with no further increment in biomass under aerobic condition. Moreover, these two enzymes introduced an alternative pathway leading toward the production of bioethanol which was more than redox-balancing steps. Acetate was accumulated as an intermediate product.
The toxic effect of acetate and oxygen limitation might be the obstacles which stopped further cell growth. This was the basic for my study to continue in the second part where I transformed second plasmid containing phaCABRE operon and vgb gene in the recombinant strain Escherichia coli BL21_pARD33. The limitation of oxygen diffusion in batch culture with time was solved by vgb gene which helped in the generation of higher biomass as the results of high oxygen availability. Subsequently, acetate was utilized as substrate by phaCAB operon in the second pathway, which converted acetyl-CoA to P3HB. In this part I found the recombinant strain Escherichia coli BL21_pARD33-BHB2 harboring the two plasmids accounted in better utilization of crude glycerol with higher production of two desirable products: bioethanol and Poly-3-hydroxybutyrate simultaneously. Despite for both products competing for the same carbon sources, this strategy was wholesome results, as their parent's strains had 2-fold lower glycerol utilization potency along with lower products yield. The maximum production of bioethanol and P3HB in the recombinant strain Escherichia coli BL21_pARD33-BHB2 were noted to be 17.4 mmol L-1 and 30.2% (w/w dry cell weight) respectively.
Finally, I investigated the stability of both the plasmids under different conditions (LB medium and define medium with and without inducers). The results showed that Escherichia coli BL21_pARD33-BHB2 had more than 50 % stability after 96 hours of post-culture. The overall strategy demonstrated above introduced a potential production manner of bioethanol as an extracellular biofuel product and P3HB as water insoluble inclusions inside Escherichia coli. This strategy can save the total production time whereas both the products can be recovered separately.
TABLE OF CONTENTS
Abstract in English..........................................................................................................................V
Abstract in Chinese..................................................................................................................V I I I
Acknowledgements.......................................................................................................................I X
Table of Contents......................................................................................................................... X I
List if tables.................................................................................................................................X V
List of figures............................................................................................................................X V I
CHAPTER 1: INTRODUCTION AND OVERVIEW
1.1 Introduction...................................................................................................................1
1.1.1 General Background.......................................................................................1
1.1.2. Current fuel....................................................................................................2
1.1.3. Alternative Energy.........................................................................................3
1.1.4. Biomass..........................................................................................................5
1.1.5. Energy from Biomass....................................................................................6
1.1.6. Biodiesel........................................................................................................7
1.1.7. Glycerol..........................................................................................................9
1.1.8. Glycerol to Value added product.................................................................11
1.1.9. Bioethanol....................................................................................................13
1.1.10. Microbial Strains........................................................................................16
1.1.11. Utilization of glycerol by E. coli................................................................18
1.2. HYPOTHESIS............................................................................................................20
1.3. OBJECTIVES.............................................................................................................20
1.4. MOTIVATIONS AND SCOPE OF THIS DISSERTATION....................................21
1.5. OUTILNES.................................................................................................................23
1.6. REFERENCES...........................................................................................................24
CHAPTER 2: CONSTRUCTION OF RECOMBINANT E. COLI FOR GLYCEROL UTILIZATION AND ETHANOL PRODUCTION
2.1. INTRODUCTION......................................................................................................27
2.1.1. Glycerol metabolism....................................................................................27
2.1.2. Aldehyde reductase......................................................................................30
2.1.3. Aldehyde dehydrogenase.............................................................................31
2.2. MATERIALS AND METHODS................................................................................34
2.2.1. Bacterial strains and plasmid used...............................................................34
2.2.1.1. E. coli BL21 (λDE3).....................................................................35
2.2.1.2. Ralstonia eutropha H16.................................................................35
2.2.1.3. Plasmid..........................................................................................36
2.2.1.4. Plasmid pBAD33..........................................................................36
2.2.2. Construction of Plasmid...............................................................................37
2.2.3. Medium used for bacterial growth...............................................................41
2.2.3.1. LB medium...................................................................................41
2.2.3.2. Minimal define medium................................................................42
2.2.3.3. Crude glycerol...............................................................................43
2.2.4. Stock preparation.........................................................................................44
2.2.5. Culture Condition.........................................................................................45
2.2.6. Plasmid Extraction.......................................................................................45
2.2.7 .Restriction digestion....................................................................................46
2.2.8. Agarose gel electrophoresis.........................................................................47
2.2.9. Assay of expression enzymes......................................................................48
2.2.10. Analytic Methods.......................................................................................49
2.2.10.1. Determination of cell concentration............................................49
2.2.10.2. Determination of products yield.................................................49
2.3. RESULTS AND DISCUSSION.................................................................................50
2.3.1. Gel electrophoresis.......................................................................................50
2.3.2. Effect of pBAD33 plasmid on crude glycerol utilization............................51
2.3.3. Impact of Alrd and AldH upon crude glycerol consumption......................52
2.3.4. Acetic acid production.................................................................................53
2.3.5. Bioethanol production..................................................................................55
2.4. CONCLUSION...........................................................................................................57
2.5. REFERENCES...........................................................................................................58
CHAPTER 3: CONSTRUCTION OF RECOMBINANT E. COLI FOR SIMULTANEOUS PRODUCTION OF P3HB AND ETHANOL FROM GLYCEROL
3.1. INTRODUCTION......................................................................................................61
3.1.1. Poly-hydroxyalkanoates...............................................................................61
3.1.2. P3HB accumulation.....................................................................................65
3.1.3 Plasmid stability............................................................................................66
3.2. MATERIALS AND METHODS................................................................................69
3.2.1. Bacterial strains and plasmid used...............................................................69
3.2.2. Competent cell preparation..........................................................................70
3.2.3. Transformation.............................................................................................70
3.2.3.1 Heat shock......................................................................................71
3.2.4. Construction of E. coli BL21_pARD33-BHB2...........................................71
3.2.5. Analytical Methods......................................................................................74
3.2.5.1. Determination of cell concentration..............................................74
3.2.5.2. Determination of products yield...................................................74
3.2.5.3. Determination of P3HB................................................................74
3.3. RESULTS AND DISCUSSION.................................................................................76
3.3.1. Effects of Alrd and AldH in E. coli strains..................................................76
3.3.2 Synthesis of products....................................................................................79
3.3.2.1. Acetic acid....................................................................................79
3.3.2.2. Bioethanol.....................................................................................79
3.3.2.3 P3HB..............................................................................................80
3.3.3. Growth of E. coli_pARD33-BHB2..............................................................82
3.3.4 Structural plasmid stability in E. coli BL21_pARD33-BHB2......................83
3.3.5 Segregational plasmid stability in E. coliBL21_pARD33-BHB2................85
3.3.6. Anaerobic growth of Different strains.........................................................87
3.4. CONCLUSION...............................................................................................89
3.5. REFERENCES...............................................................................................90
CHAPTER 4 OVERALL RESULTS AND DISCUSSION
4.1. Use of crude glycerol over pure glycerol........................................................93
4.2. Future work.....................................................................................................98
4.3. References.................................................................. ....................................99
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Chapter 4:
1. Sarma S. J., Brar S. K., Sydney E. B., Bihan Y. L., Buelna G. and Soccol C. R.: Microbial hydrogen production by bioconversion of crude glycerol: A review, Int. J Hydrogen Energ., 37, 6473-6490 (2012).
2. Dharmadi, Y., Murarka, A., and Gonzalez, R.: Anaerobic fermentation of crude glycerol by E. coli: a new platform for metabolic engineering, Biotechnol. Bioeng, 94, 821–829 (2006).
3. Durnin, G., Clomburg, J., Yeates, Z., Alvarez, P. J. J., Zygourakis, K., Campbell P., and Gonzalez R.: Understanding and harnessing the microaerobic metabolism of glycerol in Escherichia coli, Biotechnol. Bioeng., 103(1), 148–161, (2009), DOI 10.1002/bit.22246
4. Suhaimi, S. N., Phang,1 L.Y., Maeda, T., Abd-Aziz1, S., Wakisaka, M., Shirai, Y., and Hassan M. A.: Bioconversion of glycerol for bioethanol production using isolated Escherichia coli SS1, Brazilian J. Microbiol., 506–516 (2012).
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