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研究生：	王耀文 Wang, Yao-Wen
論文名稱：	脂肪分解酵素之生產、固定化並應用於生質柴油之合成 Burkholderia lipase production, immobilization and applications in biodiesel synthesis
指導教授：	張嘉修 Chang, Jo-Shu
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2010
畢業學年度：	98
語文別：	中文
論文頁數：	127
中文關鍵詞：	生質柴油、反應曲面法、酵素固定化、1,3 特異性脂肪酶
外文關鍵詞：	Biodiesel, respond surface methodology, immobilized lipase, Burkholderia sp., transesterification
相關次數：	點閱：50 下載：0
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本研究探討Burkholderia sp.脂肪分解酶之生產、固定化以及生質柴油之合成。在酵素生產方面，本研究以實驗設計法提高Burkholderia sp.之脂肪分解酶產量，所採用的實驗設計法為反應曲面法(RSM)，先以二水準因子法篩選出對酵素活性有較大影響之因子，所考慮之因素有olive oil，yeast extract和hexadecane之濃度，其結果顯示olive oil和yeast extract對酵素產量有較明顯之影響。再以此兩因素做反應曲面法分別求得最佳濃度為5.54 ml/L和2.175 g/L，其活性可以達到16.765 U/ml。為了大量生產脂肪分解酵素，本研究以發酵槽方式培養並探討其發酵策略，其探討的策略主要為通氣速率與pH 控制。實驗結果發現以通氣速率為1 vvm與pH控制在6.5的條件下，在發酵培養24小時下可得22.67 U/ml之酵素活性。
本研究接著將所生產之酵素進行酵素固定化，所探討之固定化方法為吸附法(HMP)與共價鍵結法(AMP)。結果顯示，吸附法所得到之固定化酵素，在單位載體量下有較大的酵素活性。其Langmuir isotherm等溫吸附曲線顯示以吸附法之最大的酵素吸附量為4619 U/g，而其Kd值為2.16 U/ml。本研究亦探討HMP固定化酵素(HMP-E)的再生程序與重複使用性。結果顯示，HMP-E再經過丙酮、酒精與Triton X100清洗後可以再回復其原始吸附能力；而在重複使用性方面，在連續重複操作六次後，HMP-E其初始反應速率仍能維持不變。因此，本研究所開發之固定化酵素具有高重複使用性，應有商業化應用之潛力。
最後本研究探討以固定化酵素(HMP-E)進行生質柴油合成之研究，其探討因素包括含水量、轉速、溫度、醇油比、油種、醇類、甘油影響、多階段甲醇添加、co-solvent、載體前處理與pH影響。其結果顯示含水率在10~20%較佳，溫度為40度下有較快的反應速率，醇油莫爾比可以承受到4.22:1，醇類以甲醇與乙醇有較好的反應性。本研究所開發之固定化酵素，對食用油與廢廚油的轉酯化皆有良好的催化能力；至於甘油、多階段甲醇添加、co-solvent、載體前處理與調控pH等策略則無法促進其反應速率。最後，本研究探討為何所開發的固定化酵素在生質柴油轉化率達60%以上後，其反應速率會趨於遲緩。探討方法是隨反應時間針對FAME、FFA、monoglyceride、diglycerides與triglycerides進行分析，結果發現在反應後期有10% FFA與20% monoglyceride之累積殘留，因此判斷為後期反應速率決定步驟在於esterification和acyl migration，並間接證明本實驗所使用之脂肪分解酶可能為1,3-specific lipase。為了解決後期反應速率變慢之問題，本研究以商業化lipase Novozyme 435協同本研究開發之固定化酵素進行生質柴油合成(共酵素策略)，結果發現可在18小時內即可達到達到92.5%生質柴油轉化率。

Strategies for Burkholderia lipase production, lipase immobilization, and lipase-catalyzed biodiesel production were investigated. The production of the lipase was affected by the addition of olive oil, yeast extract and hexadecane. The optimal lipase production strategy was determined with the aid of response surface methodology (RSM). According to RSM, 16.765 U/ml lipase would be produced from Burkholderia sp. as the concentrations of olive oil and yeast extract in the growth broth are 5.775 ml/L and 2.175 g/L, respectively. Using the optimal medium composition, the Burkholderia lipase was then produced in 5 L fermentor. It was found that aeration rate and pH were the most crucial factors affecting the production of lipase. The fermentor study shows that in 24 hours up to 22.675 U/ml of lipase was produced at pH 6.5 and an aeration rate of 1 vvm.
Lipase was immobilized on the ferrous oxide supports modified by hydrophobic (HMP) or amino group (AMP). Lipase was immobilized on the supports via hydrophobic interaction and the formation of covalent bonding between the lipase and the functional group on HMP and AMP. The results indicate that the hydrophobic group grafted on HMP has high affinity for lipase. Simulation with Langmuir isotherm shows that the maximum activity of HMP immobilized lipase (HMP-E) was 4619 U/g.
The regeneration and reusability of HMP-E were also studied. The used HMP was recovered by the post-treatments of acetone, ethanol and Triton X100, respectively. HMP-E was then reused to catalyze the reaction of biodiesel production. After 6 regeneration and reuse cycles, the activity of the regenerated HMP-E remained similar activity to its original one, indicating a good stability and reusability of the HMP immobilized lipase.
Next, the immobilized lipase was used for biodiesel production. The influential factors examined include water content, stirring rate, temperature, methanol-oil ratio, oils type, acyl acceptors and glycerol concentration. The results show that biodiesel production rate was enhanced when the reaction is carried at pH 6.5 and 40oC with a water content of 10-20 wt%. The best methanol to oil molar ratio for biodiesel production was 4.22:1. However, the vegetable oil type, pH and, the addition of glycerol and co-solvent did not significantly affect the production of biodiesel.
Time-course monitoring of the reaction solution composition shows accumulation of mono-glyceride (20 wt%) and free fatty acids (10 wt%). Thus, the Burkholderia lipase could be 1,3-specific lipase. The accumulation of mono-glyceride and free fatty acids could explain why the biodiesel conversion became very slow when the transesterification reached 60% conversion. With the addition of Novozyme 435, the delay in reaction rate in the late stage could be improved.

Contents
摘要	I
Abstract	IV
Acknowledgment	VI
Contents	VIII
List of Tables	XIII
List of Figures	XV
Chapter 1 Introduction	1
1 Motivation and purpose	1
Chapter 2 Literature review	3
1 Biomass energy	3
1.1 Bio-ethanol and bio-butanol	4
1.2 Hydrogen	5
1.3 Biodiesel	6
2 Biodiesel production	8
2.1 Alkaline catalysis	8
2.2 Acid catalysis	10
2.3 Lipase catalysis	12
2.4 Supercritical alcohol process	14
3 Lipase	15
4 Immobilized enzyme	22
4.1 Classification of immobilization methods	22
4.2 Immobilization of lipase	25
5 Respond surface methodology (RSM)	28
Chapter 3 Materials and methods	30
1 Chemicals, materials and strain	30
2 Equipment	32
3 Analysis and measurements	33
3.1 Preparation of emulsified substrate for pH-stat assay	33
3.2 Determination of lipase activity using pH-stat titration	33
3.3 The definition of lipase activity	33
3.4 The activity assay of immobilized lipase (HMP-E)	34
3.5-1 Analysis of biodiesel (with Stabilwax column)	34
3.5-2	Measurement of biodiesel (with Rtx-Biodiesel TG column)	………………………………………………………..36
3.6 Biodiesel production by alkaline catalysis	37
3.7 Biodiesel production by acidic catalysis	37
3.8 The estimation of the molecular weight of olive oil	38
3.9 Calculation of biodiesel conversion	38
3.10 Preparation of stock of the bacterium used in this study	38
3.11 Preparation of crude lipase powder	39
3.12 Lipase isolation via salting out	39
3.13 Dialysis	40
3.14 SDS-PAGE	40
3.15 Zymography	41
4 Lipase production	41
4.1 Lipase production in flasks	41
4.2 Two-level factorial design	42
4.3 The method of path of steepest ascent	43
4.4 Response surface methodology	44
4.5 Fermentation	46
5 Properties of the Burkholderia lipase	46
5.1 Optimum temperature for lipase hydrolysis	46
5.2 Optimum pH for lipase hydrolysis	46
5.3 Thermal stability of lipase	47
5.4 The influence of pH on lipase activity	47
6 Synthesis of enzyme immobilization supports	47
6.1 The synthesis of supporter (magnetic particles, MP)	47
6.2 Surface modification using the SMP support	48
6.3 Grafting hydrophobic group onto the surface of supporter (HMP)	48
6.4 Grafting –NH2 group onto the surface of supporter (AMP)	49
7 Lipase immobilization	49
7.1 Hydrophobic interaction adsorption	49
7.2 Immobilization by amino group modified magnetic particles	49
7.3 Adsorption curve of HMP and MP	50
7.4 Langmuir isotherm of adsorption of lipase on HMP	50
7.5 The desorption of immobilized lipase	50
7.6 HMP recovery at each step of desorption process	51
8 Transesterification	52
8.1 Biodiesel production catalyzed by immobilized lipase	52
8.2 Recycling ability of immobilized enzyme	52
8.3 Methanol inhibition experiments	52
8.4 Biodiesel produced from one-step and stepwise methanol feeding procedure	53
8.5 Addition of Co-solvent for enhancing biodiesel production	53
8.6 Effect of reaction parameters on the production of biodiesel	53
8.7 Pre-treatment of immobilized lipase particles	54
8.8 Effect of glycerol on the production of biodiesel	54
8.9 Transesterification of food oil and waste cooking oil with methanol	54
8.10 Effect of different acyl acceptors on biodiesel production	54
8.11 The effect of stabilizing pH value on biodiesel production	55
8.12 Transesterification by dual-enzyme strategy	55
Chapter 4 Results and discussion	56
1 Introduction	56
2 Effect of growth medium composition on lipase production	56
3 Medium optimization for fermentative lipase production by statistical experimental design	61
3.1 Two-level factorial design and the method of path of steepest ascent	61
3.2 Response surface methodology (RSM)	64
4 Lipase production from Burkholderia sp.	69
4.1 Effect of aeration rate on lipase production	69
4.2 Effect of initial pH on lipase production	71
5 The properties of lipase	74
6 Immobilization of Burkholderia lipase	79
6.1 Supports properties	79
6.2 Lipase immobilization	84
6.3 Langmuir adsorption isotherm	86
6.4 Hydrolysis, storage, regeneration and reusing ability of HMP	89
7 Factors affecting the reaction rate of transesterification	95
8 Strategies to improve biodiesel production by the immobilized Burkholderia lipase	100
8.1 Methanol inhibition	100
8.2 Glycerol effect	101
8.3 Pretreatment of immobilized lipase	105
8.4 Effect of free fatty acid formation	106
9 Different acyl acceptors and oils applied to biodiesel production	……………………………………………………………….. 108
10 Mechanism study of Burkholderia lipase.	111
Chapter 5 Conclusions	118
References	120
                                    

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