琥珀酸是檸檬酸循環(TCA cycle)過程中主要的中間產物之一，可應用在食品、醫藥、化妝品、表面活性劑、綠色溶劑、防腐劑和除草劑等方面，也可作為許多工業上重要化學品的前驅物，像是1,4-丁二醇，四氫呋喃等，是極具商業價值的特用化學品。由琥珀酸轉化得到的1,4-丁二醇和琥珀酸本身可用來製備具有優良熱性能與機械性能以及熱塑性加工性能的聚丁二酸丁二醇酯(PBS)，而PBS是新興的生物可降解性聚合物，有極大商機。傳統石油精煉生產琥珀酸涉及複雜的過程和昂貴的污染物處理，導致較高生產成本，因此低耗能，低污染的琥珀酸生物醱酵製程，相對的具有競爭力。尤其是面對持續增加的琥珀酸市場需求，開發具有成本效益且可永續發展的琥珀酸生產製程實為當務之急。
本研究首先將Actinobacillus succinogenes ATCC55618菌株以聚乙烯醇（PVA）進行固定化，並用於生產琥珀酸。為了進一步提升利用固定化細胞生產琥珀酸的效能，我們探討固定化細胞操作濃度、初始葡萄糖濃度和曝入的二氧化碳等對琥珀酸醱酵的影響，以獲得最適化操作條件。實驗結果發現，最適化的操作條件為15%固定化細胞操作濃度在初始葡萄糖濃度40 g/L、pH值為7及溫度37oC的條件下供給0.02 vvm的二氧化碳(45%)，其琥珀酸最大產量、產率以及產能分別為 25.5 g/L、0.875 mol/mol及3.23 g/L/h。與懸浮的細胞相比，利用固定化細胞進行發酵可以有效提升1.35倍的琥珀酸產量、1.08倍的產率和1.52倍的生產速率。此外，用30%固定化細胞在初始葡萄糖濃度40 g/L的條件下，可以獲得最大琥珀酸生產速率為5.18 g/L/h，而產量及產率分別為25.5 g/L和0.937 mol/mol。
在饋料批次琥珀酸醱酵系統中，以循環葡萄糖饋料(cyclic glucose feeding)作為饋料策略可獲得最大琥珀酸濃度(48.5 g/L)與生產速率(2.63 g/L/h)。為了降低產物抑制，在本實驗中利用離子交換樹脂結合饋料批次進行產物之同步移除。實驗結果發現，饋料批次結合產物同步移除裝置時，琥珀酸濃度能提升1.23倍，達到59.5 g/L。與饋料批次相比，產率也從0.583 g/g提升至0.699 g/g。
為了進一步提升琥珀酸生產速率，本研究嘗試以連續式生產系統進行琥珀酸醱酵，結果發現以含40 g/L葡萄糖的進料操作，在HRT為6小時時，可得到最大的生產速率為3.42±0.13 g/L/h，且此系統穩定操作時間可長達1個月。此外，為了降低醱酵成本及符合永續生產理念，本研究選用微藻生質體作為葡萄糖與酵母萃取物之替代料源。由研究成果發現，以藻類(Chlorella vulgaris ESP-31)作為料源替代葡萄糖與酵母萃取物時，可得較高的琥珀酸產率(0.720 g/g)，並且可降低76%的生產成本。與文獻相比，本研究成果頗具競爭力，並顯示微藻具有潛力取代精緻糖質作為料源用於琥珀酸發酵生產，可降低生產成本以利於商業運轉之可行性，以達成永續經營之最終目標。

Succinic acid is one of the intermediates of the tricarboxylic acid (TCA) cycle. Succinic acid is potentially used in food, pharmaceutics, cosmetics, surfactants, green solvents, antiseptics and herbicides, also as a precursor of many industrially important chemicals including 1,4-butanediol, tetrahydrofuran, and so on. A new biodegradable polymer, poly(butylene succinate) (PBS) with excellent thermal and mechanical properties as well as thermoplastic process ability, can be produced by succinic acid and 1,4-butanediol converted from succinic acid. Conventional succinic acid production is based on fossil oil refining, which involves complicate process and expensive pollutant treatment, leading to a high production cost. In light of this, producing succinic acid from fermentation seems to have the niche due to its merits of low pollution, low energy consumption, and sustainability. Considering the increasing growth in the market for succinic acid, it is of great demand to develop more sustainable and cost-effective succinic acid producing process.
In this study, succinic acid was produced by microbial fermentation, using polyvinyl alcohol (PVA) immobilized cells of a succinic acid-producing bacterium Actinobacillus succinogenes ATCC55618. To further enhance succinic acid production with immobilized A. succinogenes ATCC55618, the PVA particle loading, initial glucose concentration and carbon dioxide supply were optimized in batch fermentation. The experimental results show that the optimal conditions are: PVA particle loading, 15%; pH, 7.0; temperature, 37oC; carbon dioxide supply, 45%/0.02 vvm; glucose concentration, 40 g/L, This results in a succinic acid concentration, productivity and yield of 25.5 g/L, 3.23 g/L/h and 0.875 mol/mol, respectively. Compared with the suspended cells, the succinic acid production, yield, and productivity with PVA-immobilized cell fermentation were significantly improved by 1.35, 1.08, 1.52 times, respectively. In addition, using 30% w/v PVA particle loading with initial glucose concentration of 40 g/L, the batch culture could obtain a maximum productivity of 5.18 g/L/h with a yield and concentration of 0.937 mol/mol and 25.5 g/L, respectively.
In the fed-batch succinic acid fermentation with cyclic glucose concentration feeding, the maximum succinic acid concentration was 48.5 g/L and the productivity was 2.63 g/L/h. To alleviate product inhibition encountered in fed-batch fermentation, the fed-batch process was coupled with ion exchange resin to successfully remove succinic acid from fermentation broth. The succinic acid concentration improved by 1.23 fold as it reached 59.5 g/L and the succinic acid yield was improved from 0.583 g/g to 0.699 g/g, compared with fed batch fermentation without removal of succinic acid.
To develop more efficient and cost-effective process for succinic acid production, continuous fermentation strategies were examined. Continuous production of succinic acid with immobilized cell fermenter was stably operated for a long time (about one month), giving a had maximum productivity of 3.42±0.13 g/L/h with 6 h HRT and glucose utilization of 87.9±2.46% (glucose concentration in the feed was 40 g/L).
The microalgae biomass as renewable feedstock was used to overcome the challenge of high production cost. The microalgae biomass as renewable feedstock on separate hydrolysis fermentation (SHF) as carbon source and nitrogen source without yeast extract had maximum yield of 0.720 g/g and also reduced 75.8% of its production cost. High performance production of succinic acid with immobilized cells using microalgal biomass as renewable feedstock by SHF has the potential to replace glucose for industrial production.

中文摘要 I
Abstract III
Acknowledgement V
Contents VII
List of Tables XI
List of Figures XIII
Chapter 1 INTRODUCTION 1
1.1 Motivation and Purpose 1
1.2 Research Scheme 4
Chapter 2 LITERATYRE REVIEW 6
2.1 Succinic Acid Fermentation Technologies 6
2.1.1 Metabolic Pathway for Succinic Acid Production 8
2.1.2 Fermentation strategy 10
2.1.3 pH 12
2.1.4 CO2 14
2.2 Immobilization of Cells 15
2.3 Separation Technology for Succinic acid Recovery 17
2.4 Sustainable Renewable Feedstock 21
Chapter 3 MATERIALS AND METHODS 25
3.1 Chemicals and Materials 25
3.2 Equipment 26
3.3 Microorganisms and Culture Medium 27
3.3.1 Actinobacillus succinogenes ATCC55618 27
3.3.2 Microalgae used (Chlorella vulgaris ESP-31) 29
3.4 Analytical Assays 31
3.4.1 Determination of Soluble Component Concentration 31
3.4.2 Determination of Cell Concentration 36
3.4.3 Measurement of gaseous products 36
3.4.4 Analysis of transient behavior by modified Gompertz equation 37
3.4.5 Characterization of Immobilized Cells by Scanning Electronic Microscope (SEM) 38
3.4.6 Determination of carbohydrates in microalgal biomass 39
3.4.7 Determination of lipid content in the microalgal hydrolysate 39
3.4.8 Determination of amino acid content in the microalgal hydrolysate and fermentation broth 40
3.4.9 Determination of ionic compounds in the microalgal hydrolysate and fermentation broth 41
3.4.10 Determination of hydroxymethylfurfural (HMF) and furfural in the microalgal hydrolysate and fermentation broth 41
3.5 Experimental Methods 41
3.5.1 Suspension cell 41
3.5.2 Immobilized cell 42
3.5.3 Fed-batch strategy using PVA immobilized Actinobacillus succinogenes ATCC55618 for succinic acid fermentation 48
3.5.4 Effect of resin capacity 48
3.5.5 Recovery of succinic acid via ion exchange resin from fermentation broth of fed-batch culture using immobilized cells of Actinobacillus succinogenes ATCC55618 49
3.5.6 Continuous fermentation 50
3.5.7 Using microalgae as raw material for continuous succinic acid fermentation with PVA-immobilized Actinobacillus succinogenes 50
Chapter 4 RESULTS AND DISCUSSION 52
4.1 Succinic acid production by immobilization of Actinobacillus succinogenes ATCC55618 with Polyvinyl Alcohol (PVA) in batch mode 52
4.1.1 Characterization of Immobilized Cell by Scanning Electronic Microscope (SEM) 52
4.1.2 Comparison of immobilized cell fermentation with suspended cell fermentation 53
4.1.3 The fermentation performance without pH control 54
4.1.4 Effect of particle loading on succinic acid fermentation 56
4.1.5 Effect of glucose concentration on succinic acid fermentation 57
4.1.6 Effect of CO2 flow rate on succinic acid fermentation 59
4.1.7 Effect of dilution of yeast extract concentration on succinic acid fermentation 60
4.1.8 Effect of biotin on succinic acid fermentation 61
4.1.9 Effect of monosodium glutamate on succinic acid fermentation 62
4.1.10 Effect of CO2 content on succinic acid fermentation 64
4.2 Enhancing production of succinic acid by Actinobacillus succinogenes ATCC55618 on fed-batch fermentation process 75
4.2.1 Effect of fed-batch strategy on succinic acid fermentation 75
4.2.2 Effect of resin capacity 76
4.2.3 Fed-batch succinic acid fermentation with cyclic glucose feeding coupled with product recovery using ion exchange resin 77
4.3 Continuous succinic acid production by PVA-immobilized Actinobacillus succinogenes ATCC55618 81
4.3.1 Effect of HRT on succinic acid fermentation 81
4.4 Using microalgae hydrolysate as renewable raw material via SHF process on succinic acid production 85
4.4.1 Succinic acid production using microalgae as feedstock in batch mode 85
4.4.2 Effect of replacement of yeast extract with microalgae hydrolysate 87
Chapter 5 CONCLUSION 95
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