표제지
목차
국문초록 14
1. 서론 16
가. 생물반응기의 모니터링 16
1) 중·대형 생물반응기의 모니터링 17
2) 소형 생물반응기의 모니터링 18
가) 진탕배양기 19
나) 마이크로플레이트 기반 생물반응기 20
나. 형광기반 센서 22
1) 광학 센서 22
2) 형광기반 센서 23
가) 용존산소(dO₂) 센서 23
나) 수소이온(pH) 센서 27
다) 용존이산화탄소(dCO₂) 센서 29
다. 형광 센서막 31
1) 용존산소 검출용 센서막 31
가) 고분자 기반 센서막 32
나) 졸-겔 기반 센서막 34
2) 수소이온 검출용 센서막 34
가) 고분자 기반 센서막 35
나) 졸-겔 기반 센서막 36
라. 연구목적 37
마. 참고문헌 38
2. 형광검출 시스템 54
가. 형광의 발생 및 검출 54
나. 형광검출 시스템의 구성 60
1) 시스템의 구조 60
2) 광원 62
3) 광학 필터 64
4) 광 검출기 66
다. 형광검출 시스템의 특성 67
1) 광학 필터의 신호 대 잡음비 67
2) Ru(dpp)32+및 HPTS용액의 형광세기 측정(이미지참조) 68
3) LED 종류에 따른 시스템의 성능 70
라. 다채널 소형 생물반응기 시스템의 제어 및 전자회로 72
마. 요약 75
바. 참고문헌 76
3. 마이크로플레이트 기반 생물반응기 시스템의 특성 78
가. 교반 시스템에 따른 산소전달 특성 79
1) 발효액의 교반 79
2) 미생물의 산소전달 속도 80
3) 산소전달계수 82
4) 교반속도와 방해판 형태에 따른 산소전달 실험 84
가) 재료 및 방법 84
나) 결과 및 고찰 86
나. 온도분포 특성 88
1) 재료 및 방법 89
2) 결과 및 고찰 92
가) 세라믹 히터를 이용한 온도제어 92
나) 면상발열체를 이용한 온도제어 94
다. 요약 96
라. 참고 문헌 97
4. 형광센서막의 제조 및 특성 100
가. 형광센서막의 구성 100
나. 형광염료에 의한 dO₂, PH 및 dCO₂의 검출 101
1) 형광염료를 이용한 dO₂의 검출 101
2) 형광염료를 이용한 pH의 검출 102
3) 형광염료를 이용한 dCO₂의 검출 103
다. 형광센서막의 제조 및 특성 104
1) 시약 및 재료 104
2) 용존산소 검출용 센서막 104
가) 루테늄(II) 복합체의 광학특성 104
나) 센서막의 제조 106
다) 결과 및 고찰 107
3) 수소이온 검출용센서막 112
가) HPTS의 형광 특성 112
나) 센서막의 제조 114
다) 결과 및 고찰 115
4) 용존이산화탄소 검출용 센서막 118
가) 이산화탄소 검출용 형광염료 118
나) 센서막의 제조 119
다) 결과 및 고찰 120
라. 요약 127
마. 참고문헌 128
5. 마이크로플레이트 기반 생물반응기의 미생물 발효에의 응용 133
가. 실험 방법 135
1) 재료 및 방법 135
가) 생물반응기 135
나) 미생물 및 배지 137
나. 생물반응기의 성능 137
1) 교반속도에 따른 미생물 발효 특성 138
2) 온도에 따른 미생물 발효 특성 139
다. 용존산소의 온라인 모니터링 141
1) E.coli DH5a 발효공정에서 용존산소의 온라인 모니터링 142
2) Bacillus cereus 318 발효공정에서 용존산소의 온라인 모니터링 144
라. pH의 온라인 모니터링 146
1) E.coli DH5a의 발효공정에서 pH의 온라인 모니터링 148
2) B.cereus 318 발효공정에서 PH의 온라인 모니터링 150
마. 용존이산화탄소의 온라인 모니터링 154
바. 세포농도의 온라인 모니터링 156
1) E.coli DH5a 발효공정에서 세포농도의 온라인 모니터링 156
2) B.cereus 318 발효공정에서 세포농도의 온라인 모니터링 158
사. 용존산소, pH 및 세포농도의 동시 온라인 모니터링 160
1) E.coli DH5a 발효공정에서 용존산소, pH 및 세포농도의 동시 온라인 모니터링 161
2) B.cereus 318 발효공정에서 용존산소, pH 및 세포농도의 동시 온라인 모니터링 164
아. 요약 166
자. 참고문헌 168
6. 마이크로플레이트 기반 소형 생물반응기를 이용한 발효배지의 최적화 170
가. 통계적 최적화 기법 172
1) 일시일원 실시법(One factor at a time method) 172
2) 요인배치법(Factorial design method) 172
3) 플라킷-버만 설계법(Plackett-Burman design method) 173
4) 반응표면법(Response surface method) 173
나. 재료 및 방법 174
1) 균주 및 배지 174
2) 발효 실험 174
다. 결과 및 고찰 177
1) E.coli DH5a의 배지 최적화 177
2) B.cereus 318의 배지 최적화 179
라. 요약 182
마. 참고문헌 183
7. 결론 186
부록 189
가. 다채널 형광검출기 189
나. LED housing 190
다. 전자회로도 191
1) 증폭회로 191
2) 마이크로 컨트롤러 192
라. 마이크로 컨트롤러 프로그램 193
Abstract 197
감사의 글 200
Table 1-1. Fluorescence indicators and supporting matrices for oxygen sensors. 26
Table 1-2. Fluorescence indicators and supporting matrices for pH sensors. 28
Table 1-3. Fluorescence indicators and supporting matrices for dissolved carbon dioxide sensors. 30
Table 1-4. Properties of some polymers for optical oxygen sensing membrane. 32
Table 3-1. Typical respiration rates of microorganisms and cells in culture. 82
Table 3-2. Comparison of KLa values determined on the basis of the glucose oxidation with different reciprocating agitation speeds at 25℃.(이미지참조) 88
Table 4-1. Amount of NaCl to adjust the total ionic strength of the stock solution A and B. (stock solution A(1.3799 g/L of NaH₂PO₄·H₂O), B(1.799 g/L of NaHPO₄·2H₂O)) 108
Table 6-1. Medium composition in E.coli DH5a cultivation for the medium optimization experiments. 175
Table 6-2. Medium composition in Bacillus cereus 318 cultivation for the medium optimization experiments. 176
Figure 1-1. Illustration of the trade off in information output versus high-throughput capability that currently exists for various cell cultivation devices at different scales. 19
Figure 1-2. Principle of optical oxygen detection by a fluorophore. 25
Figure 2-1. The mechanism of fluorescence. The initial excitation takes place between states of same multiplicity and in accord with the Franck-Condon principle. (E: energy, S0: ground state, S₁: excitation state,...(이미지참조) 55
Figure 2-2. Plot of brightness (ε*φ) vs the wavelength of maximum absorption (λmax) for some fluorophores. (ε: extinction coefficient, φ: quantum yield)(이미지참조) 57
Figure 2-3. Set-up of optical components for the fluorescence detection. 60
Figure 2-4. Set-up of fluorescence detection system. 61
Figure 2-5. Spectral output of light from a xenon lamp and Nd:YAG laser. The "relative output" axis is scaled arbitrarily for the two light sources. 62
Figure 2-6. Electroluminescence spectrum of LEDs. The emission peaks were about 410 nm, 470 nm and 650 nm, with FWHM of 14.5 nm, 23.6 nm and 25 nm. 64
Figure 2-7. Transmission spectra of two bandpass filters, BP-524-O and LW-590. 66
Figure 2-8. Set-up of a measurement device for the ratio of signal to noise (S/NR). 68
Figure 2-9. Fluorescence intensity of Ru(dpp)32+ and HPTS solution.(이미지참조) 69
Figure 2-10. Effects of system temperature on the fluorescence detection system with different Ru(dpp)32+ and HPTS solutions.(이미지참조) 70
Figure 2-11. Fluorescence intensity of different Ru(dpp)32+ and HPTS solutions with 3 mm LED and 5 mm LED(이미지참조) 71
Figure 2-12. Function of a micro-controller. 73
Figure 2-13. Flowchart of control and measurement units with a micro-controller. 74
Figure 3-1. Shaking system of the 24-well microtiter plate bioreactor. 80
Figure 3-2. Schematic diagram of a 24-well microtiter plate with different baffles. 85
Figure 3-3. Different baffle configurations in a 24-well microtiter plate. 85
Figure 3-4. Overall oxygen transfer rate (OTR) with shaking speeds for different types of baffles (A~I). 87
Figure 3-5. Relations between reciprocating agitation speed and shaking speed unit. 88
Figure 3-6. Different types of temperature-control systems for the microplate-based bioreactor with optical on-line monitoring system (MABOOMS) (R:multichannel microbioreactor, H:ceramic heater, PH:planer... 91
Figure 3-7. Temperature measurement in the chamber of the microplate-based bioreactor system. 92
Figure 3-8. Temperature distribution of the microplate-based bioreactor system using a ceramic heater. The temperature was controlled to 35 ℃. 94
Figure 3-9. Temperature distribution of the mlcroplate-based bioreactor using a planer type heater. The temperature was controlled to 35 ℃. 95
Figure 4-1. Dynamic luminescence quenching of the excited state of the Ru(dpp)32+(R) by molecular oxygen.(이미지참조) 102
Figure 4-2. Chemical formula of Ru(dpp)32+.(이미지참조) 105
Figure 4-3. 2D-fluorescence spectra of Ru(dpp)32+ with (A):0 % dissolved oxygen and (B):100 % dissolved oxygen.(이미지참조) 106
Figure 4-4. Schematic illustration of the optical dissolved oxygen sensing membrane on a well of 24-well microtiter plate. 107
Figure 4-5. Effects of pH on the dissolved oxygen sensing membrane. 109
Figure 4-6. Fluorescence intensity of the dissolved oxygen sensing membrane with ionic strength. 110
Figure 4-7. Relative fluorescence intensity of 0 % dissolved oxygen solution with the dissolved oxygen sensing membrane at different temperatures. Fluorescence intensity at 25 ℃ was set to 100 % as reference value. 111
Figure 4-8. Stability of the dissolved oxygen sensing membrane for three months. 112
Figure 4-9. The chemistry and spectroscopy of a HPTS pH fluorescence dye. (A) Structures of the neutral and ionized forms of HPTS are shown reacting reversibly with a solution containing protons. (B) The pH dependent... 113
Figure 4-10. 2D-fluorescence spectra of HPTS in the pH buffer solutions with (a) pH 4.0 and (b) pH 8.0. 114
Figure 4-11. Calibration curve of the pH sensing membrane at various pH solutions. 116
Figure 4-12. (a) Fluorescence intensity of a pH sensing membrane at different pH value and ionic strengths. (b) Effect of temperature on the pH sensing membrane. 117
Figure 4-13. Storage stability of a pH sensing membrane for three months. 118
Figure 4-14. (a) The Construction of a CO₂ Sensing film and (b) the reaction process between HPTS and CO₂ in the fluorescence principle for CO₂ sensing. 120
Figure 4-15. (a) Fluorescence intensity at ex 450 nm/ em 515 nm and (b) fluorescence spectra of the dCO₂ sensing membrane with various dissolved carbon dioxide solution. 122
Figure 4-16. Relative fluorescence intensity with different pH values. Fluorescence intensity at pH 7 was set to 100 % as reference value. 123
Figure 4-17. Fluorescence intensity of the dissolved carbon dioxide sensing membrane with different ionic strengths. 124
Figure 4-18. Effect of temperature on the dissolved carbon dioxide sensing membrane. 125
Figure 4-19. Storage stability of the dissolved carbon dioxide sensing membrane. 126
Figure 5-1. Schematic set-up of the MABOOMS. The microplate-based bioreactor is placed inside the MABOOMS and the chamber is closed. Four optical fiber bundle carry four different wavelengths of light to the bottom... 136
Figure 5-2. (a) Effect of reciprocating agitation speed in the MABOOMS on the growth of E.coli DM5a (30 ℃). (b) Comparison of cell growths in the shaking incubator(37 ℃, 180 rpm) and the MABOOMS. 139
Figure 5-3. Optical cell density of E.coli DH5a in the MABOOMS and the shaking incubator under different temperatures. (SI: shaking incubator, MBB: 24-well microplate-based bioreactor) 140
Figure 5-4. Calibration curve of dissolved oxygen sensing membrane at various dissolved oxygen concentrations. The calibration curve was validated with a commercial oxygen electrode(Thermo Co., USA). 142
Figure 5-5. Online monitoring of dissolved oxygen concentrations in E.coli DH5a cultivation with the MABOOMS. (■: fluorescence intensity, -: dissolved oxygen) 144
Figure 5-6. Online monitoring of dissolved oxygen concentrations in B.cereus 318 cultivation with the MABOOMS. (■: fluorescence intensity, -: dissolved oxygen) 146
Figure 5-7. Calibration curves of the pH sensing membranes at various pH buffer solutions. 147
Figure 5-8. Online monitoring of pH values in E.coli DH5a cultivation with the MABOOMS. (Row 1, 3, 5: E.coli DH5a cultivation, Row 2, 4, 6: blank sample without E.coli DH5a cultivation, ■: fluorescence intensity, -: PH) 149
Figure 5-9. Offline PH values in E.coli DH5a cultivation with the MABOOMS, the shaking incubator and the bioreactor. (MMB: microplate-based bioreactor, SI: shaking incubator, BR: bioreactor) 150
Figure 5-10. Online monitoring of pH values in B.cereus 318 cultivation with the MABOOMS. (Row 1, 3, 5: B.cereus 318 cultivation, Row 2, 4, 6: blank sample without B.cereus 318 cultivation, ■: fluorescence intensity, -: pH) 152
Figure 5-11. Offline pH values in B.cereus 318 cultivation with the MABOOMS, the shaking incubator and the bioreactor. (MBB: microplate-based bioreactor, SI: shaking incubator) 153
Figure 5-12. Online monitoring of dissolved carbon dioxide concentrations in E.coli DH5a and B.cereus 318 cultivations with the MABOOMS. 155
Figure 5-13. Online monitoring of reflectance(RF) at 650 nm and normalized reflectance(norm. RF=I0-I I0) in E.coli DH5a cultivation with the MABOOMS. (I0: reflectance at initial time, I: reflectance at a time, -: reflectance 650 nm,...(이미지참조) 157
Figure 5-14. Offline measurement of the absorbance at 600 nm (i.e. optical density) in E.coli DH5a cultivation with the MABOOMS, shaking incubator and the bioreactor. (MBB: microplate-based bioreactor, SI: shaking... 158
Figure 5-15. Online monitoring of reflectance(RF) at 650 nm and normalized reflectance(norm. RF=I0-I I0) in B.cereus 318 cultivation with the MABOOMS. (I0: reflectance at initial time, I: reflectance at a time, -: reflectance 650 nm,...(이미지참조) 159
Figure 5-16. Offline measurement of the absorbance at 600 nm (i.e. optical density) in B.cereus 318 cultivation with the MABOOMS, the shaking incubator and the bioreactor. (MBB: microplate-based bioreactor, SI:... 160
Figure 5-17. Schematic diagram of a 24-well microplate-based bioreactor with 4-divided sensing membranes. 161
Figure 5-18. Online monitoring of fluorescence intensity and reflectance for dO₂, pH and cell mass with the 4-divided sensing membranes in E.coli DH5a cultivation with the MABOOMS. (-: dO2, ■:norm. RF, ○: pH) 163
Figure 5-19. Online monitoring of fluorescence intensity and reflectance for dO₂, PH and cell mass with the 4-divided sensing membranes in B.cereus 318 cultivation with the MABOOMS. (-: dO2, ■:norm. RF, ○: pH) 165
Figure 6-1. Comparison between the shaking incubator and the MABOOMS for medium optimization. 172
Figure 6-2. Online monitoring of fluorescence intensity and reflectance for dO₂, pH and cell mass in E.coli DH5a cultivation with various medium components for the medium optimization using the MABOOMS. (■: dO2, -:... 178
Figure 6-3. Online monitoring of fluorescence intensity and reflectance for dO₂, pH and RF in B.cereus 318 cultivation with various medium components for the medium optimization using the MABOOMS. (■: dO2, -:norm. RF, ○: pH) 181