臺灣博碩士論文加值系統

本研究以Saccharomyces cerevisiae進行濃醪酒精發酵程序，並開發出發酵培養基。以~107 (cell ml-1)的酵母菌接菌量、1 % (w/v)玉米浸漬粉末 (corn steep powder, CSP)、以及其他培養基的組成維持在低添加量時，在48小時內可達到較高的酒精產出量 (158.27±4.29 g l-1, 等同於 20.06±0.54 %, v/v)。相對於較低酒精產出量 (138.08±7.63 g l-1, 等同於 17.50±0.97 %, v/v)，是由~106 (cell ml-1)的酵母菌接菌量、5 % (w/v) CSP，條件下所得到的結果。當酵母菌添加維持高添加量時，可提高葡萄糖消耗速率；但是CSP添加為高添加量時，將減緩酵母菌的增殖、以及在發酵末期酒精生產量低。
“t50“為logistic dynamic model所推導得到的結果，期望能夠透過此數學模式以及利用多種不同培養基組成的培養，來論證濃醪酒精發酵之情況。“ “為酵母菌消耗一半量的葡萄糖所需時間，以及” ”為酒精產出總量的一半所需時間。一個24階層實驗設計，其因子為尿素、硫酸氨、玉米浸漬粉、以及酵母菌接種量，利用此多個不同培養基的配方來證明“t50“可區別濃醪酒精發酵培養基之效力。由分析的結果顯示“t50”可以用來區分不同的培養基的培養結果，並且透過16組實驗的數據分析，區分出四大組，此四大組為CSP添加及酵母接菌量所排列組合而成的。由兩組不同的濃醪酒精發酵培養基，所獲得的“t50”結果(8.34~9.12 小時)，選擇恰當的發酵培養基，將可縮短發酵時間，同時增加酒精的年產量。
以22實驗規劃做為探討游離氮源添加對甜高粱濃醪酒精發酵之影響。單純以高粱進行發酵時，在發酵初期迅速且完全的攝取有限的可利用游離氮源。以添加及不添加游離氮源，進行同步糖化發酵時，添加游離氮源之酒精產率為3.03 g L-1h-1 (等同於在48小時產出146 g l-1酒精)；當不添加游離氮源時，其酒精產率為0.93 g L-1h-1 (等同於在156小時產出144 g l-1酒精)。反之，進行先糖化後發酵 (SHF)時，添加游離氮源時酒精產率為2.18 g L-1h-1 (等同於在68小時產出139 g l-1酒精)；當不添加游離氮源時，其酒精產率為0.68 g L-1h-1 (等同於在185小時產出125 g l-1酒精)。由結果顯示，進行同步糖化發酵 (SSF)，並且添加游離氮源時，將獲得較有效率之發酵程序，意味著擁有較高的酒精產率以及較短的發酵時間。本實驗與過去相似的研究互相比較後，發現本研究的酒精產能提升了60%。

In this study, we report the development of a fermentation medium designed specifically for very-high-gravity (VHG) ethanol production by Saccharomyces cerevisiae. By keeping amount of yeast pitched at ~107 cells ml-1, corn steep powder (CSP) at 1 % (w/v), and other medium components at low levels, the highest ethanol concentration (158.27±4.29 g l-1, equivalent to 20.06±0.54 %, v/v) was attained in 48h using batch fermentation. Comparatively, the lowest ethanol concentration (138.08±7.63 g l-1, equivalent to 17.50±0.97 %, v/v) was obtained when amount of yeast pitched and CSP was maintained at ~106 cells ml-1 and 5% w/v, respectively. A high-level amount of yeast pitched gives rise to fast glucose consumption rate; however, a high-level CSP retards yeast propagation, resulting in low ethanol production rate in the later stage of fermentation.
t50, derived from logistic dynamic model, was proposed and implemented to evaluate VHG fermentation subjected to the variation of different culture compositions. t50 is the time required to consume 50% of initial glucose concentration (symbolized as ), or is the time required to produce half of the final ethanol concentration (symbolized as ). A 24 factorial experimental design, including four key ingredients: urea, ammonium sulfate, corn steep powder, and amount of yeast pitched, was planned to attest the applicability of t50 to discriminate the effectiveness of different growth media during VHG fermentation. The analysis of variance shows that t50 could differentiate among different VHG growth media, and be further adopted to cluster 16 datasets into four groups according to corn steep powder and amount of yeast pitched. The t50 difference between two VHG growth media varies 8.34 ~9.12 h, implying that the selection of a proper growth medium for VHG fermentation could drastically reduce fermentation time, thereby increasing the annual ethanol productivity.
A 22 experimental plan was implemented to investigate the effect of dual free amino nitrogen (FAN) supplementation on the sweet sorghum fermentation under very-high-gravity (VHG) conditions. A rapid initial FAN uptake rate signified insufficient provision of utilizable nitrogen by sorghum mash alone. The ethanol productivity was 3.03 g L-1h-1 (equivalent to 146 g l-1 ethanol in 48 h when FAN was added) as compared to 0.93 g L-1h-1 (equivalent to 144 g l-1 ethanol in 156 h when FAN was not added) by using configuration of simultaneous saccharification and fermentation (SSF). Whereas, the ethanol productivity was 2.18 g L-1h-1 (equivalent to 139 g l-1 ethanol in 64 h when FAN was provided) as compared to 0.68 g L-1h-1 (equivalent to 125 g l-1 ethanol in 185 h when FAN was not provided) by using configuration of separate hydrolysis and fermentation (SHF). Results indicate that SSF configuration with a dual addition of FAN-containing substances is the most efficient fermentation configuration among investigated cases. Such a configuration results in the highest ethanol yield and the shortest fermentation time. The ethanol productivity of this configuration was increased by 60 % as compared to other similar findings reported previously.

TABLE OF CONTENTS
ENGLISH ABSTRACT I
CHINESE ABSTRACT IV
TABLE OF CONTENTS VI
LIST OF FIGURES XI
LIST OF TABLE XIV
NOMENCLATURE XVII
CHAPTER I. Introduction 1
1.1. Background 1
1.1.1. The application of Ethanol 1
1.1.2. Biofuel 2
1.1.3. Ethanol production microorganism 5
1.1.4. The advantage and disadvantage of very high gravity ethanol fermentation technology 7
1.1.5. Separate hydrolysis and fermentation & Simultaneous saccharification and fermentation 11
1.1.6. Composition of ethanol fermentation medium 14
1.1.6.1. Carbohydrate 15
1.1.6.2. Nitrogen 22
1.1.6.3. Other nutrient 28
1.1.7. Kinetic model 29
1.1.8. The potential for red sorghum grains as carbon source in ethanol fermentation process 32
1.1.9. The enzyme for starch liquefy process and saccharify process 33
1.2. Motivation 35
1.3. Scope of the study 37
CHAPTER II. Materials and Methods 39
2.1. Materials 39
2.1.1. Microorganism 39
2.1.2. Chemical 40
2.1.3. Enzyme 43
2.1.4. Instruments 44
2.2. Method 46
2.2.1. Cultivation and Stock 46
2.2.2. Seed culture 47
2.2.3. The mineral salts composition and prepare 48
2.2.4. The composition of stock vitamin solution 49
2.2.5. The free amino nitrogen measurement 50
2.2.6. Ethanol fermentation 52
2.2.6.1. For “Development of growth media for very-high-gravity ethanol fermenmtation by Saccharomyces cerevisiae.” 52
2.2.6.2. For “On the development of t50 for evaluating very-high-gravity fermentation.” 54
2.2.6.3. For “The effect of fermentation configurations and FAN supplementation on ethanol production from sorghum grains under very-high-gravity conditions.” 55
2.2.6.3.1. Preparation of sorghum mash 55
2.2.6.3.2. Ethanol fermentation using sorghum mash as carbon source 56
2.2.6.3.3. Experimental plan 57
2.2.7. Fermentation broth samples treatment 58
2.2.8. High pressure liquid chromatography (HPLC) 59
2.2.9. Gas chromatography 60
2.2.10. Glucose analysis 61
2.2.11. Viable and dead cell count 62
2.2.12. Data analysis for “On the development of t50 for evaluating very-high-gravity fermentation.” 63
CHAPTER III. Results and Discussion 65
3.1. Development of growth media for very-high-gravity ethanol fermentation by Saccharomyces cerevisiae 65
3.1.1. Amount of yeast pitched and corn steep powder 75
3.1.2. Yeast cell population profile 78
3.1.3. Free amino nitrogen utilization profile 81
3.1.4. Glucose consumption profile 84
3.1.5. Ethanol production profile 87
3.1.6. Discussion 89
3.2. On the development of t50 for evaluating very-high-gravity fermentation 95
3.2.1. Overview 95
3.2.2. Ethanol and glucose concentration profiles 96
3.2.3. The statistical analysis 99
3.2.4. The correlation and with nutrient supplement 105
3.2.5. Different period glucose consumption rate 107
3.2.6. Different period ethanol production rate 109
3.2.7. Discussion 112
3.3. The effect of fermentation configurations and FAN supplementation on ethanol production from sorghum grains under very-high-gravity conditions 116
3.3.1. Glucose consumption profile 116
3.3.2. Ethanol production profile 119
3.3.3. FAN utilization profile and viable cell profile 122
3.3.4. Discussion 126
CHAPTER IV. Conclusions 129
CHAPTER V. Recommendation for Future Studies 131
CHAPTER VI. References 132

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