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研究生：	洪子琁 Hong, Zih-Syuan
論文名稱：	利用微藻料源進行高效能固定化細胞發酵以生產乳酸 High Performance Production of Lactic Acid with Immobilized Cells Using Microalgal Biomass as Renewable Feedstock
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：	2016
畢業學年度：	104
語文別：	英文
論文頁數：	98
中文關鍵詞：	聚乳酸、Lactobacillus plantarum 23 、乳酸醱酵、聚乙烯醇-固定化細胞、微藻
外文關鍵詞：	poly-lactic acid, Lactobacillus plantarum 23, lactic acid fermentation, microalgae, Poly Vinyl Alcohol (PVA)-immobilized cell
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近年來大量使用由石化原料所製成的不可降解性塑膠對環境造成很大的衝擊，且過度消耗石化資源也引發嚴重的能源危機，為了解決此問題，生物可降解性塑膠逐漸被受矚目，生物可分解原料-聚乳酸也因此成為全球注目的焦點。乳酸的應用範圍相當廣泛除了應用於食品、醫藥、香粧品以及化學工業等，其中最為重要的應用乃作為聚乳酸的原料，因此全球對乳酸的需求相當迫切，乳酸的來源可由微生物發酵與化學合成所生產，但相較化學合成法之下以發酵法生產乳酸生產成本較高，因此提高乳酸生產效率與降低乳酸發酵的生產成本為主要的挑戰。
本研究利用本土菌株Lactobacillus plantarum 23以懸浮菌體相進行乳酸批次發酵。由研究成果可知，批次試驗最佳的培養條件為厭氧、pH 5.5與葡萄糖濃度40 g/l，其乳酸產量、產率與生產速率分別為26.46 g/l、0.66 g/g與4.28 g/l/h。為了提升乳酸發酵效率，本研究採取固定化細胞策略，其乳酸濃度、乳酸產率與乳酸生產速率與懸浮菌體比較分別提升了1.43倍、1.39倍與1.12倍，而最佳的固定化細胞系統之操作條件為包埋菌體濃度5.25 g/l與固定化細胞濃度為 12.5%。在碳源測試方面，本研究選用木醣與蔗糖進行乳酸發酵，實驗結果顯示選用蔗糖其生產速率可達到6.39 g/l/h。在培養基質方面，為了降低培養基質成本並提高產率，本研究以不同稀釋倍率的培養基質進行對乳酸生產，實驗結果顯示當培養基質稀釋為50%，有最佳的乳酸生產效率。
此外，本研究也採取兩種碳源進料策略進行乳酸生產，分別為控制剩餘葡萄糖濃度(residual glucose concentration control)與循環葡萄糖饋料(cyclic glucose feeding)，結果以cyclic glucose feeding饋料策略可得較高乳酸產量為108 g/l。為了降低乳酸抑制現象，本研究利用離子交換樹脂結合饋料批次進行同步移除，其結果顯示結合產物同步移除裝置能提升饋料批次乳酸生產效率。
為了進一步提升乳酸生產速率，本研究嘗試以連續式生產系統進行乳酸生產，結果發現在HRT為4小時與固定化細胞濃度為12.5%，其乳酸濃度、產率、生產速率分別為23.17 g/l、0.90 g/g與5.79 g/l/h。當固定化細胞濃度增加為50%，其乳酸濃度、產率、生產速率分別為31.75 g/l、0.93 g/g與7.94 g/l/h，由此可見增加固定化細胞有利於提升反應速率與產量，且此系統穩定操作時間可長達3個月。最後，為了降低基質成本，本研究選用微藻作為料源，利用4wt.%的硫酸進行酸水解反應，將微藻內之碳水化合物轉化成還原糖進行連續式乳酸生產，由研究成果發現，相較於以相同濃度的葡萄糖進行乳酸醱酵，以藻類為料源可得較高的乳酸產率且達到理論值的99%，乳酸生產速率也提升至9.93 g/l/h。此結果顯示選用微藻當作料源不但可以降低乳酸發酵的生產成本，也可以提升整體乳酸發酵的效率。因此，微藻是非常具有潛力取代精緻糖質料源且可邁向低成本高永續性的乳酸生產製程之開發。

In recent years, extensive usage of fossil-fuel-based plastics has left an undesirable impact on the environment and caused accelerated global energy depletion. Microbial production of bio-plastics is being looked upon as the best alternative, since both the production process and the end product are eco-friendly.
Lactic acid has received increasing attention all over the world, due to its widespread applications in cosmetic, food, pharmaceutical and chemical industries. In particular, lactic acid has been extensively used for the synthesis of bio-based plastics, acting as the monomer of polylactic acid (PLA), which is the most promising biodegradable and environmentally friendly material on market. Lactic acid can be produced by microbial fermentation, but higher production costs are always a concern. Enhancement of lactic acid production efficiency and reduction of substrate costs are important issues that needs to be addressed to produce lactic acid by microbial fermentation.
In this study, the Lactobacillus plantarum 23 was used to produce lactic acid. The cells were used as suspended cells and the experiment was conducted in batch mode. Anaerobic culture conditions, pH value of 5.5 and glucose concentration of 40 g/l were selected as the optimal conditions and the lactate production, yield, and productivity were 26.46 g/l, 0.66 g/g, and 4.28 g/l/h, respectively. The strategy of immobilized cells was used to enhance lactic acid fermentation by Lb. plantarum 23. Lactic acid production (37.93 g/l), yield (0.91 g/g), and productivity (4.96 g/l/h) were enhanced 1.43, 1.39, and 1.12 times, respectively, compared with fermentation using suspended cells. A cell concentration of 5.25 g/l in Poly Vinyl Alcohol (PVA) for immobilization and particle loading of 12.5% were selected as optimal condition for PVA-immobilized cells fermentation. Carbon sources other than glucose, like xylose and sucrose were tested for lactic acid production in batch fermentation using PVA immobilized cells. The results indicated that using sucrose as carbon source gives high productivity (6.39 g/l/h) compared to other sugars. To reduce the cost of modified MRS medium and enhance the rate of lactic acid fermentation, the use of diluted modified MRS medium was investigated. 50% dilution of modified MRS medium had comparable productivity with the undiluted medium and was decided as optimum medium composition.
Two fed-batch strategies were used for lactic acid production, including residual glucose concentration control and cyclic glucose concentration feeding. The lactic acid produced from immobilized cells by cyclic glucose concentration feeding could produce higher lactic acid concentration of 108 g/l. In-situ removal of lactic acid by ion-exchange resin could alleviate the problem of end-product inhibition and enhance the rate of fermentation and conversion of glucose to lactic acid.
Continuous lactic acid production was conducted to achieve higher productivity. Continuous production with particle loading of 12.5% and HRT of 4 h had lactic acid production of 23.17 g/l, yield of 0.90 g/g, and productivity of 5.79 g/l/h. Increasing the particle loading from 12.5% to 50 % had higher production of 31.75 g/l, yield of 0.93 g/g and productivity of 7.94 g/l/h with glucose consumption of 96.62%. Continuous production was efficiently improved by 50% of particle loading and could be stably operated for more than three months. It is important that lactic acid has to be produced from cheaper feedstock to reduce substrate costs of fermentation. In this direction, microalgal biomass based reducing sugars were used for lactic acid fermentation. The lactic acid yield was close to the theoretical yield of 99%, and productivity was enhanced to 9.93 g/l/h. The results showed that microalgae biomass as feedstock is advantageous, because of reduced production cost, enhanced lactic acid fermentation and it has the potential to replace refined sugars in such fermentation processes.

Contents
中文摘要	I
Abstract	III
Acknowledgement	VI
Contents	VIII
List of Tables	XII
List of Figures	XIV
Chapter 1	INTRODUCTION	1
1	Motivation and Purpose	1
2	Research Scheme	4
Chapter 2	LITERATURE REVIEW	6
1 Lactic Acid Fermentation Technologies	6
1.1 Metabolic Pathway for Lactic Acid Production	6
1.2 Fermentation strategy	10
1.3 pH	11
2 Immobilization of Cells	12
3 Separation Technology for Lactic Acid Recovery	14
4 Microalgae as Sustainable Renewable Feedstock	16
Chapter 3	MATERIALS AND METHODS	18
1 Chemicals and Materials	18
2 Equipment	20
3 Microorganisms and Mediums	22
3.1 Lactobacillus plantarum 23	22
3.2 Chlorella vulgaris ESP-31	22
3.3 Medium Composition for Lactobacillus plantarum 23	23
3.4 Medium Composition for the cultivation of Chlorella vulgaris ESP-31	24
3.5 The composition of Chlorella vulgaris ESP-31 microalgal hydrolysate	25
4 Analytical Assays	25
4.1 Determination of Soluble Component Concentration	25
4.2 Determination of Optical Purity	26
4.3 Determination of Cell Concentration	26
4.4 Measurement of carbohydrates content in microalgal hydrolysate	26
4.5 Characterization of Cell Immobilization by Scanning Electronic Microscope (SEM)	27
4.6. Determination of lipid content in the microalgal hydrolysate	27
4.7 Determination of amino acid content in the microalgal hydrolysate and fermentation broth	28
4.8 Determination of ionic compound in the microalgal hydrolysate and fermentation broth	28
4.9 Determination of hydroxymethylfurfural (HMF) and furfural in the microalgal hydrolysate and fermentation broth	29
5 Experimental Methods	29
5.1 Suspension cell	29
5.2 Immobilized cell	31
5.3 Fed-batch strategy using PVA immobilized Lactobacillus plantarum 23 for lactic acid fermentation	34
5.4 Recovery of lactic acid via ion exchange resin from fermentation broth of fed-batch culture using immobilized cells of Lactobacillus plantarum 23	35
5.5 Continuous fermentation	36
5.6 Using microalgae as raw material for continuous lactic acid fermentation with PVA-immobilized Lactobacillus plantarum 23	37
Chapter 4	RESULTS AND DISCUSSION	38
1 Lactic acid production using suspended cell in batch mode	38
1.1 Effect of culture type on lactic acid fermentation	38
1.2 Effect of pH-control on lactic acid fermentation	41
1.3 Effect of glucose concentration on lactic acid fermentation	44
1.4 Kinetics of lactic acid production with Lactobacillus plantarum 23	45
2 Lactic acid production by immobilization of Lactobacillus plantarum 23 with Polyvinyl Alcohol (PVA) in batch mode	46
2.1 In comparison immobilized cell fermentation with suspended cell fermentation	46
2.2 Effect of cell concentration in PVA on lactic acid fermentation	48
2.3 Effect of particle loading on lactic acid fermentation	51
2.4 Effect of carbon source on lactic acid fermentation	52
2.5 Using microalgae hydrolysate as renewable raw material via SHF process on lactic acid production	56
2.6 Effect of dilution of modified MRS medium composition concentration on lactic acid fermentation	64
3 Enhancing production of lactic acid by Lactobacillus plantarum 23 on fed-batch fermentation process	67
3.1 Effect of fed-batch strategy on lactic acid fermentation	67
3.2 Fed-batch lactic acid fermentation with cyclic glucose feeding coupled with product recovery using ion exchange resin	71
4 Continuous lactic acid production by PVA-immobilized Lactobacillus plantarum 23	78
4.1 Effect of HRT on lactic acid fermentation	78
4.2 Effect of particle loading on lactic acid fermentation	80
5 Continuous lactic acid production using microalgae as feedstock	87
Chapter 5  CONCLUSION	90
REFERENCES	92

 
 

                                    

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