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研究生：	謝勤毅 Hsieh, Chin-I
論文名稱：	生物界面活性劑表面素重複發酵之生物反應器設計與操作策略 Bioreactor design and operation strategies for repeated surfactin fermentation
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：	2007
畢業學年度：	95
語文別：	英文
論文頁數：	82
中文關鍵詞：	改良式生物反應器、活性碳、固定化細胞、乳膠共聚物、表面素、脂胜肽、生物界面活性劑
外文關鍵詞：	polyurethane-polyurea copolymer, bioreactor design, cell immobilization, surfactin, lipopeptide, activated carbon, biosurfactant
相關次數：	點閱：109 下載：1
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本研究使用Bacillus subtilis ATCC 21332進行生物界面活性劑表面素(surfactin)重複操作生產潛力之評估。其所生產出之脂胜肽類生物界面活性劑－表面素具有優越之表面活性，為目前最具效果之生物界面活性劑之一，並有令人期待之商業應用價值，但是由於其生產價格偏高，影響實際應用之可行性。有鑑於此，本實驗室致力於發展更有效率且更經濟之表面素醱酵技術，以降低其商業化應用之門檻。
本實驗室於先前的研究已經由反應器的改良以及固體載體的使用，在批次發酵上得到理想的結果。目前致力於進一步將生產流程朝向重複操作邁進。本研究首先採用的是固定化細胞生產的方式進行重複批次生產實驗。結果顯示，採用添加活性炭載體之乳膠共聚物（polyurethane- polyurea copolymer）固定化菌株生產surfactin，可生產約1,340 mg/L之表面素，且固定化顆粒方便於重複批次之操作。此外，以設置外接循環式活性炭卡匣系統之改良式生物反應器進行懸浮菌重複操作培養時，若採用再接種(re-inoculation)之策略，其前兩次重複操作皆可得優異的表面素產量，其表面素濃度可達6000 mg/L。

Bacillus subtilis ATCC 21332 was used to produce surfactin via repeated operations. Surfactin, a lipopeptide biosurfactant, is recognized as one of the most effective biosurfactant available and possesses promising commercial applications. This motivated us to develop viable fermentation technology to produce surfactin in a more efficient way in order to lower the high production cost. In our recent studies, excellent surfactin producing performance was achieved in batch fermentation from innovative bioreactor design and addition of solid carrier as stimulator for surfactin production. In the present study, strategies enabling repeated operation for surfactin production were developed. The first attempt was using immobilized cell systems. Of the immobilized-cell system examined, PU (polyurethane- polyurea copolymer) immobilized cells supplemented with activated carbon resulted in the best performance with a surfactin concentration of 1,340 mg/L, and the particles were easy to be operated repeatedly. On the other hand, repeated operation with suspended-cell system was also attempted using innovative bioreactor design, in which an outer circulation activated carbon cassette system was equipped. This bioreactor setup allowed production of a high surfactin concentration of 6000 mg/L in the first two repeated batches.

Contents
Acknowledgement……………………………………………………………..i
Abstract (Chinese)……………………………………………………………Ⅰ
Abstract (English)………………………………………………………….....II 
Contents………………………………………………………………………III
List of Figures……………………………………………………………….VI
List of Tables………………………………………………………………...IX
Notations……………………………………………………………………...X
Chapter 1 Introduction………………………………………………………...1
	1.1 Motivation and purpose………………………………………………1
	1.2 Outline………………………………………………………………..2
Chapter 2 Literature review…………………………………………………...5
2.1 Surfactants……………………………………………………………5
2.2 Biosurfactants………………………………………………………...9
2.3 Surfactin……………………………………………………………..17
2.4 Characterizations of surfactin……………………………………….19
	2.4.1 Intrinsic characteristics……………………………………….19
	2.4.2 Foaming property……………………………………………..19
	2.4.3 Surface activity and hemolytic activity……………………....20
	2.4.4 Activity of oil displacement…………………………………..21
	2.4.5 Hydrophobicity……………………………………………….22
	2.4.6 Surface mobility………………………………………………22
	2.4.7 Membrane solubilization……………………………………..22
2.5 Surfactin production and purification……………………………….24
		2.5.1 Medium compositions and fermentative conditions………….24
		2.5.2 Bioreactor designs……………………………………………27
Chapter 3 Materials and Methods…………………………………………...32
	3.1 Chemicals and materials…………………………………………….32
	3.2 Equipments………………………………………………………….34
	3.3 Microorganisms, medium and cultivation…………………………..37
	3.4 Surfactin production with immobilization cells (flask tests)….…….38
	3.5 Fermentor experiments……………………………………………...39
		3.5.1 Bioreactor design……………………………………………..39
		3.5.2 Fermentation conditions for surfactin production……………40
		3.5.3 Solid carrier…………………………………………………..40
       3.5.4 Fed-batch surfactin fermentation……………………………..41
3.5.5 Repeated batch surfactin fermentation……………………….41
	3.6 Analytical methods and surfactin purification………………………44
3.6.1 Determination of cell concentration………………………….44
		3.6.2 Measurement of ammonium-nitrogen concentration…………44
3.6.3 Measurement of glucose concentration………………………44
		3.6.4 Surfactin analysis with HPLC………………………………..45
		3.6.5 Isolation and purification of surfactin………………………...46
Chapter 4 Repeated batch assessment of surfactin production using immobilized-cell system………………………………………….47
4.1 Introduction…..………………………………………………………47
4.2 Surfactin production with original PU immobilized cells……………48
4.3 Supplemental materials………………………………………………49
4.4 Effect of cell loading in immobilized 
cells on surfactin production…………………………………………51
4.5 Repeated batch operation using immobilized cells…………………..53
Chapter 5 Bioreactor design and operation strategies for 
repeated surfactin fermentation…………………………………..54
5.1 Introduction……………………………………………………..........54
5.2 Surfactin fermentation using original bioreactor…………………….54
5.3 Surfactin fermentation using the modified bioreactor………………..61
     5.3.1 Modification of bioreactor and batch
          fermentation tests…………………………………………...61
     5.3.2 Fill-and-draw operation with modified bioreactor………......62
5.4 Effect of surfactin concentration on surfactin fermentation……….....66
5.5 Effect of ammonia-nitrogen concentration 
on surfactin fermentation……………………………………………..69
5.6 Repeated operation with seed culture re-inoculation………………...71
Chapter 6 Conclusions……………………………………………………….73
References………………………………………………………...................74

List of Figures

Figure 1.1 A flowchart describing the research scope in this study…………..4
Figure 2.1 Basic structure of a surfactant……………………………………..5
Figure 2.2 Nonionic surfactant polyoxyethylene (20) sorbitan 
monooleate (Tween 80 or T80) ………………………...………….6
Figure 2.3 Sodium dioctylsulphosuccinate…………………………………...6
Figure 2.4 Hexadecylsulphonic acid………………………………………….7
Figure 2.5 Dodecylamine hydrochloride……………………………………...7
Figure 2.6 Dodecylpyridinium chloride……………………………………....7
Figure 2.7 Zwitterionic form of N-dodecyl-N,N-dimethyl betaine…………...7
Figure 2.8 Structure of surfactin………………………………………………8
Figure 2.9 Relationship between surfactant concentration and critical 
micelle concentration…………………………………………….9
Figure 2.10 Rhamnolipids from Pseudomonas aeruginos..............................11
Figure 2.11 Sophorose lipid from Torulopsis apicola.....................................11
Figure 2.12 Surfactin from Bacillus subtilis………………………………...12
Figure 2.13 Lichenysin from Bacillus licheniformis………………………...12
Figure 2.14 Iturin A from Bacillus subtilis…………………………………..12
Figure 2.15 Biosurfactant produced by Corynebacterium lepus, 
where R1, R2 = alkyl, X = hydrogen, ethylamine, inositol……...13                 
Figure 2.16 Emulsan from Acinetobacter calcoaceticus…………………….13
Figure 2.17 Structure of surfactin; n = 7, 8, or 9…………………………….18
Figure 2.18 A diagram of surfactin production regulated 
by two quorum sensing systems in Bacillus subtilis…………….18
Figure 2.19 Apparatus used in semi-batch foam fractionation………………29
Figure 2.20 Apparatus used for the simultaneous growth and 
collection of the culture foam…………………………………..29
Figure 2.21 Scheme of airlift bioreactor…………………………………….30
Figure 2.22 Schematic description of the modified bioreactor module……..30
Figure 3.1 Immobilized Bacillus subtilis ATCC 21332 cells beads………...39
Figure 3.2 The photograph of the modified bioreactor system……………...42
Figure 3.3 Cylindrical activated carbon with a diameter of 
3-4 mm and a height of 9 mm…………………………………...43
Figure 3.4 The CAC was an indispensable factor in this research…………..43
Figure 3.5 Calibration line of glucose concentration………………………..45
Figure 3.6 Calibration line of surfactin concentration………………………46
Figure 4.1 Surfactin production with original PU immobilized cells……….48
Figure 4.2 Surfactin production in PU immobilized cells 
supplemented with chitosan, sugar and AC 
powder as additives……………………………………………...50
Figure 4.3 Surfactin production with PU immobilized cells 
supplemented with different amount of activated 
carbon powder…………………………………………………...51
Figure 4.4 Effect of cell loading in PU immobilized 
cells on surfactin production…………………………………….52
Figure 4.5 Repeated operation of surfactin production by 
PU immobilized cells supplemented with activated 
carbon carriers…………………………………………………...53
Figure 5.1 Surfactin concentration and cell-growth profile 
from original bioreactor design with 
carrier-supplemented strategy……………………………………56
Figure 5.2 Fed-batch fermentation for surfactin production………………...57
Figure 5.3 Cumulative surfactin production by fed-batch fermentation…….58
Figure 5.4 Surfactin fermentation with fill-and-draw operation…………….60
Figure 5.5 Batch surfactin fermentation with modified bioreactor………….63
Figure 5.6 Surfactin production with fill and draw operation 
using the modified bioreactor……………………………………65
Figure 5.7 Test for finding damage caused by high 
residual surfactin concentration….................................................67
Figure 5.8 Repeated operation eliminating product inhibition………………68
Figure 5.9   Repeated operation with reduced ammonia-nitrogen concentration…………………………………………………….70
Figure 5.10 Repeated surfactin fermentation using the 
seed culture re-inoculation strategy……………………………72







List of Tables

Table 2.1 Microorganisms and corresponding biosurfactants……………….16
Table 2.2	Comparison of surfactin producing performance between 
this work and comparable studies…………………………………31
Table 3.1 The composition of LB medium………………………………….37
Table 3.2 The composition of MSI medium………………………………...37
Table 5.1 Comparison of surfactin production performance 
between the present study (batch fermentation) 
and literature……………………………………………………..64
                                    

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