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系統識別號 U0026-0309201312233100
論文名稱(中文) 以Klebsiella sp. Zmd30 進行二醇類之發酵製程開發
論文名稱(英文) Developing fermentation process for diols production using Klebsiella sp. Zmd30
校院名稱 成功大學
系所名稱(中) 化學工程學系碩博士班
系所名稱(英) Department of Chemical Engineering
學年度 101
學期 2
出版年 102
研究生(中文) 翁巧玲
研究生(英文) Chiao-Ling Wong
學號 N38971130
學位類別 博士
語文別 英文
論文頁數 141頁
口試委員 指導教授-張嘉修
口試委員-吳文騰
口試委員-李篤中
口試委員-陳博彥
口試委員-魏毓宏
口試委員-黃介辰
口試委員-黃良銘
口試委員-顏宏偉
口試委員-王翔郁
中文關鍵字 1,3-丙二醇  2,3-丁二醇  固定化細胞  回應曲面法  粗甘油  農業廢棄物  醱酵策略 
英文關鍵字 Klebsiella sp.  1,3-propanediol  2,3-butanediol  response surface methodology  crude glycerol  agriculture wastes  fermentation strategies 
學科別分類
中文摘要 摘要
在本研究中從北台灣宜蘭礁溪溫泉中篩選出一株菌株Klebsiella sp. Zmd30來進行二醇類的生產,首先探討不同甘油濃度於細胞生長與細胞休眠(resting cells)時對於1,3-丙二醇 (1,3-PDO)生產速率與產率的影響。從結果中發現最佳的甘油濃度為50 g/l,而1,3-丙二醇的濃度、生產速率與產率分別為18 g/l、0.55 g/l/h與45%。相較之下,進行休眠細胞實驗時,由於缺乏氮源濃度導致細胞生長受抑制,反而造成1,3-丙二醇產率之提升。接著以固定化細胞改善細胞的穩定度與再利用性,該固定化細胞可以連續穩定操作五次,並維持相近的生產效果。此外,該固定化細胞可直接利用粗甘油進行1,3-丙二醇之生產,其生產效果與使用純甘油時相似。
本研究另一個目標是開發低成本與高效能2,3-丁二醇(2,3-BDO)之生產程序。首先以回應曲面法 (Response surface methodology, RSM)進行培養基對於2,3-丁二醇生產最適化之研究,經由RSM分析之結果可得最佳培養基配方如下:葡萄糖146 g/l、尿素2.28 g/l、MnCl2•4H2O 7.01 x 10-4 g/l、H3BO31.49 x 10-4 g/l與CoCl2•6H2O 5.70 x 10-3 g/l。於此最佳培養基條件下,其2,3-丁二醇的生產速率為1.15 g/l/h。為了降低生產成本,本研究選擇以農業廢棄物(蔗渣與稻桿)當作料源進行2,3-丁二醇之生產,先將蔗渣與稻桿進行酵素水解,再以水解液進行2,3-丁二醇之醱酵生產。結果發現,以稻桿為料源之效果優於蔗渣,其生產速率與產率分別為2.08 g/l/h與62%。
接著探討溫度與溶氧對於2,3-丁二醇生產之影響,溫度的操作條件於25 - 40 oC時,並沒有很明顯的影響。對於溶氧的部分則針對攪拌與通氣進行考量,並以RSM找出最佳操作條件,其結果為在轉速100 rpm 與通氣1vvm下有較佳的結果,其濃度、生產速率與產率分別為57 g/l、1.67 g/l/h與83%。最後探討pH值對於2,3-丁二醇生產之影響,其操作條件為pH 4.5-9.0,發現pH值為6.0時對於2,3-丁二醇生產有較佳的效果,其濃度、生產速率與產率分別為52 g/l、2.10 g/l/h與74%。
此外,本研究亦針對不同之醱酵策略進行探討,包含批次、饋料批次與連續式操作。於批次操作時,2,3-丁二醇之濃度、生產速率與產率分別為57 g/l、1.59 g/l/h與82%。而在饋料批次時,可達最大濃度為110 g/l,但是其生產速率卻低於批次培養,只有0.88 g/l/h,而產率高達94%。於連續式操作時,當其水力停留時間 (HRT)為12 hr時,可達最高生產速率2.81 g/l/h,但是其濃度只達到19 g/l,而產率達到60%。從結果來看,以饋料批次程序生產2,3-丁二醇對於商業化而言是較適合的,因為饋料批次程序能達到較高的2,3-丁二醇濃度與較高的產率,可減少下游程序之負擔與原料及操作成本之消耗。
英文摘要 Abstract
In this study, the strain Klebsiella sp. Zmd30 isolated from Chiao-Hi’s hot spring in Northern Taiwan was used for diols production. Firstly, the effect of different glycerol concentrations on 1,3-propanediol (1,3-PDO) productivity and the yield of suspended cells by Klebsiella sp. Zmd30 under growing-cell or resting-cell (nutrient-limiting) conditions. The best glycerol concentration for 1,3-PDO production was 50 g/l and the 1,3-PDO concentration, productivity, and yield were 18 g/l, 0.55 g/l/h and 45%, respectively. Using immobilized cells of Zmd30 greatly improved the operational stability and reusability of the cells, as the immobilized cells could be used for 5 cycles without significant activity loss. In contrast, cultivation on nutrient-deficient resting-cell medium was favorable for 1,3-PDO production since cell growth was limited, resulting in the increase in 1,3-PDO yield. The immobilized cells were also able to directly utilize non-pretreated crude glycerol obtained from a local biodiesel manufacturing plant for 1,3-PDO production. The 1,3-PDO production efficiency from using crude glycerol was comparable to that obtained from using pure glycerol.
Another aim of this study was to develop a low-cost and high-efficiency process for 2,3-butanediol (2,3-BDO) production. Firstly, the medium composition for the growth of the Zmd30 strain was optimized using the response surface methodology (RSM) to enhance 2,3-BDO production. Based on the RSM analysis, the optimal composition of glucose, urea and MnCl2•4H2O, H3BO3 and CoCl2•6H2O was 146, 2.28, 7.01 x 10-4, 1.49 x 10-4 and 5.70 x 10-3 g/l, respectively. With this optimal medium, the 2,3-BDO productivity was improved to 1.15 g/l/h. To reduce the production cost, agriculture wastes (i.e. bagasse and rice straw) were used as feedstock for 2,3-BDO production. It was found that using hydrolyzed rice straw resulted in better 2,3-BDO productivity than using hydrolyzed bagasse. When using rice straw as feedstock, the highest 2,3-BDO productivity and yield were 2.08 g/l/h and 62%, respectively.
Next, the effects of temperature and dissolved oxygen on 2,3-BDO production were investigated. The results show that the temperature in the range of 25 to 40 oC had no obvious effects on 2,3-BDO production. In contrast, the dissolved oxygen providing by adjusting aeration and agitation rate significantly affect the performance of 2,3-BDO production. Optimization of aeration and agitation rates was carried out using RSM, and the optimal aeration rate and agitation rate determined by RSM analysis was 100 rpm and 1 vvm, respectively. Under this optimal conditions, the 2,3-BDO concentration, productivity and yield could reach57 g/l, 1.67 g/l/h and 83%, respectively. Finally, the influences of pH control at different levels (from 4.5 to 9.0) on the distribution of metabolic products were examined. The optimal pH for 2,3-BDO production was suggested to be 6.0, at which the 2,3-BDO concentration, productivity and yield were 52 g/l, 2.10 g/l/h and 74%, respectively.
Furthermore, different fermentation strategies of batch, fed-batch and continuous operations were performed. In batch fermentation, the concentration, productivity and yield of 2,3-BDO were 57 g/l, 1.59 g/l/h and 82%, respectively. In the fed-batch operation, much higher 2,3-BDO concentration (about 110 g/l) and yield (94%) was obtained, but the productivity (0.88 g/l/h) was lower when compared to that obtained from batch operation. In the continuous culture operated at a HRT of 12 h, the highest 2, 3-BDO productivity was markedly increased to 2.81 g/l/h, while the 2, 3-BDO concentrations dropped to only 19 g/l and the yield was 60%. The results suggest that the fed-batch operation might be more suitable for applying on the commercialized 2,3-BDO producing process due to the high 2,3-BDO titer and yield.
論文目次 目錄
摘要………………………………………………………………………I
Chapter 1 Introduction..............................1
1.1 Motivation and purpose............................1
1.2 Research scope of this dissertation...............3
Chapter 2 Literature review.........................9
2.1 Glycerol..........................................9
2.1.1 Bioconversion of glycerol by industrial microbiology.............................................10
2.2 Lignocellulose....................................11
2.2.1 Rice straw.......................................13
2.2.2 Bagasse..........................................13
2.3 Pretreament of lignocelluloses materials.........14
2.3.1 Chemical pretreatment............................16
2.3.2 Biological pretreatment..........................17
2.3.3 Enzymatic pretreatment...........................18
2.3.4 Physical pretreatment............................19
2.3.5 Physico-chemical pretreatment....................21
2.4 Production of 1,3-propanediol....................24
2.4.1 Properties of 1,3-propanediol....................25
2.4.2 Chemical synthesis of 1,3-propanediol............25
2.4.3 Microbial formation of 1,3-propanediol...........28
2.5 Production of 2,3-butanediol.....................29
2.5.1 Properties of 2,3-butanediol.....................30
2.5.2 Synthesis of 2,3-butanediol......................31
Chapter 3 Material and Methods.....................33
3.1 Chemical and material............................33
3.2 Equipment........................................36
3.3 Bacterial strain and cultivation medium..........38
3.3.1 Diols-producing bacteria and culture medium......38
3.3.1.1 Culture medium for 1,3-PDO production............40
3.3.1.2 Culture medium for 2,3-BDO production............41
3.4 Analysis methods.................................43
3.4.1 Determination of cell concentration..............43
3.4.2 Determination cellulose and hemicelluloses concentration............................................43
3.4.3 Determination of reducing sugar concentration by DNS method...............................................43
3.4.4 Determination of soluble products by high performance liquid chromatography (HPLC).................44
3.4.5 Determination of endoglucanase activity..........45
3.4.6 Determination of b-glucosidase activity..........45
3.4.7 Determination of FPase activity..................46
3.4.8 Determination of glycerol dehydratase (GDHt) activity.................................................46
3.5 Cell immobilization..............................47
3.6 Response surface methodology (RSM) analysis......47
3.7 Measurement of kinetic parameters.............49
Chapter 4 Characterization and optimization of 1,3-propanediol fermentation with Klebsiella sp. Zmd30 using suspended resting-cells............................50
4.1 Selection and identification of strains for 1,3-PDO production...............................................50
4.2 Effect of glycerol concentration on 1,3-PDO production by suspended cells under cell-growth conditions...............................................54
4.3 Production of 1,3-PDO by suspended cells under resting-cell (nutrient-limiting) conditions..............56
4.4 Effect of crude glycerol sources on 1,3-PDO production...............................................66
4.5 Summary..........................................68
Chapter 5 Fermentation strategies for 1,3-porpanediol production from Klebsiella sp. Zmd30 by immobilized cells under resting-cell conditions............................69
5.1 Production of 1,3-PDO by immobilized cells under resting-cell (nutrient-limiting) conditions..............69
5.2 Converting crude glycerol to 1,3-PDO using immobilized cells under resting-cell (nutrient-limiting) conditions...............................................75
5.3 Summary..........................................76
Chapter 6 Optimizing medium composition and culture conditions for 2,3-butanediol fermentation with Klebsiella sp. Zmd30................................................78
6.1 Selection and identification of strains for 2,3-butanediol production................................79
6.2 Effect of glucose concentration on 2,3-butanediol production by Klebsiella sp. Zmd30.......................80
6.3 Effect of the nitrogen source and concentration on 2,3-butanediol production by Klebsiella sp. Zmd30........82
6.4 Effect of trace elements on 2,3-butanediol production by Klebsiella sp. Zmd30.......................84
6.5 Response surface analysis for optimal medium compositions on 2,3-butanediol production by Klebsiella sp. Zmd30....................................................89
6.6 Hydrolysis of agriculture waste for 2,3-butanediol production by Klebsiella sp. Zmd30.......................96
6.7 Summary.........................................101
Chapter 7 Optimization of fermentation conditions for 2,3-butanediol production with Klebsiella sp. Zmd30.....102
7.1 The effect of aeration rate for 2,3-butanediol production by Klebsiella sp.............................102
7.2 The effect of agitation speed for 2,3-butanediol production by Klebsiella sp.............................103
7.3 The effects of temperature on 2,3-butanediol production by Klebsiella sp.............................105
7.4 Response surface methodology for optimization of cultivation conditions for 2,3-butanediol production with Klebsiella sp. Zmd30....................................107
7.5 The effects of pH on 2,3-butanediol production by Klebsiella sp. Zmd30....................................112
7.6 Summary.........................................114
Chapter 8 Effect of fermentation strategies on 2,3-butanediol production with Klebsiella sp. Zmd30.....116
8.1 Batch fermentation for 2,3-BDO production with Klebsiella sp. Zmd30....................................119
8.2 Fed-Batch fermentation for 2,3-BDO production with Klebsiella sp. Zmd30....................................120
8.3 Production of 2,3-BDO using continuous fermentation with Klebsiella sp. Zmd30...............................124
8.4 Effect of fermentation mode on 2,3-BDO production performance with Klebsiella sp. Zmd30...................126
8.5 Summary.........................................126
Chapter 9 Conclusions.............................128
References..............................................130

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