[표제지 등]

제출문

요약문

Summary

그림목차

표목차

목차

제1장 미호기성 Bacillus 속 분리 세균의 반연속 배양에 의한 수소 생성 19

제1절 서론 19

제2절 실험 재료 및 방법 21

1. 실험재료 21

가. 사용 균주 21

나. 배지 및 균주 보존 21

2. 실험방법 21

가. 회분 배양 21

(1) 환원성 배지 조제 21

(2) 배양 방법 21

나. 반연속 배양 24

(1) 반연속 배양계의 설계 24

(2) 배양 방법 24

다. 균체량의 측정 24

라. 수용성 환원당량 정량법 24

마. 수소 생성량 측정법 29

바. 유기용매와 유기산의 정량법 31

제3절 실험 결과 및 고찰 32

1. Bacillus sp. I-17, I-19의 회분 배양에 의한 수소 생성 32

2. Bacillus sp. I-17, I-19의 증식 곡선과 비증식 속도 35

3. Bacillus sp. I-17, I-19의 반연속 배양에 의한 수소 생성 35

4. Bacillus sp. I-7의 반연속 배양계에서의 glucose 농도의 영향 47

5. Bacillus sp. I-17의 수소 생성에 미치는 배양 조건의 검토 53

가. 온도의 영향 53

나. 교반 속도의 영향 55

다. 초기 접종 세포 농도의 영향 55

6. Bacillus sp. I-17의 증식과 수소 생성에 영향을 미치는 배지 조건의 검토 55

가. Yeast extract와 Peptone의 영향 58

나. Cysteine·HCl, Vitamin (Biotin, p-Aminobenzoic acid)의 영향 60

다. 무기 성분의 영향 60

라. 반연속 배양계에서 공급해주는 Yeast extract와 Peptone, 무기 성분의 적정량의 검토 63

7. Bacillus sp. I-17의 glutamate에 의한 수소 생성 63

제4절 결론 69

제5절 참고문헌 71

제2장 유기발효폐액을 이용한 바이오수소의 생성과 페액처리효과 77

제1절 서론 77

제2절 실험 재료 및 방법 81

1. 실험 재료 81

가. 사용 균주 81

나. 배지 및 균주 보존 81

2. 실험 방법 84

가. 환원성 배지의 조제 84

나. 배양 방법 84

다. 휴면 세포 현탁액(Resting cell suspension)의 준비 87

라. 균체량의 측정 87

마. Starch의 정량 87

바. 수소 생성량 측정법 88

사. 유기산과 유기 용매의 정량법 89

아. DO 측정법 89

자. BOD 측정법 91

제3절 실험 결과 및 고찰 92

1. 각 수소 생성균의 유기물질로부터의 수소 생성 92

가. Clostridium butyricum NCIB 9576의 유기물질로부터의 수소 생성 92

(1) 여러가지 당류로부터의 수소 생성 92

(2) 여러가지 아미노산으로부터의 수소 생성 94

나. Rhodopseudomonas sp.E 15-1 과 R. sphaeroides K-7의 유기물질로부터의 수소 생성 94

(1) 여러가지 당류로부터의 수소 생성 94

(2) 여러가지 아미노산으로부터의 수소 생성 94

다. Cl.butyricum NCIB 9576과 R.sphaeroides K-7의 혼합배양시의 유기물질로부터의 수소 생성 98

(1) 여러가지 당류로부터의 수소 생성 98

(2) 여러가지 유기산으로부터의 수소 생성 98

(3) 여러가지 아미노산으로부터의 수소 생성 102

(4) glucose, starch, lactose, sucrose에서의 수소 생성 102

(5) 각 농도별 starch에서의 수소 생성 108

2. 각 수소 생성균의 L-glutamate농도에 따른 수소 생성 115

3. 유기 폐자원을 이용한 수소 생성과 폐액의 처리 정도 124

가. Bacillus 속과 Clostridium 속 세균의 유기폐액의 농도에 따른 수소 생성 124

나. 광합성 세균과 혼합배양에서의 유기 폐액의 농도에 따른 수소 생성 126

다. 혼합 배양에서 유기 폐액에 첨가한 여러가지 당류로부터의 수소 생성 126

라. 혼합배양에서 유기 폐액에 첨가한 여러가지 유기산으로부터의 수소 생성 126

마. 혼합배양에서 유기 폐액에 첨가한 starch, lactose, maltose로 부터의 수소 생성 129

바. 혼합배양에서 유기 폐액을 이용한 배지에 vitamin solution첨가의 필요성 검토 131

제4절 결론 135

제5절 참고문헌 137

제3장 바이오수소 생성에 대한 총관적인 결론 145

Table 1.1. Composition of modified PYG medium 22

Table 1.2. Operating evolution of the semi-continuous culture system. 27

Table 1.3. Hydrogen evolution of Bacillus sp. I-17 at various rate of replacement in semi-continuous feeding 48

Table 1.4. Hydrogen evolution of Bacillus sp. I-19 at various replacement rate in semi-continuous feeding 49

Table 1.5. Effect of yeast extract and peptone on growth and hydrogen evolution of Bacillus sp. I-17 59

Table 1.6. Effects of inorganic components in PYG medium on growth and hydrogen evolution of Bacillus sp. I-17 62

Table 1.7. Effect of yeast extract and peptone, and inorganic components concentration on growth and hydrogen evolution of Bacillus sp. I-17 in semi-continuous culture 65

Table 2.1. Composition of modified PYG medium 82

Table 2.2. Composition of Modified Ormerod medium 83

Table 2.3. Composition of organic waste water 85

Table 2.4. Effect of various carbohydrates on hydrogen evolution and evolution rate by Cl.butyricum NCIB 9576 93

Table 2.5. Effect of various amino acids on hydrogen evolution and evolution rate by Clostridium butyricum NCIB 9576 95

Table 2.6. Effect of various carbohydrates on hydrogen evolution and by Rhodopseudomonas sp. E 15-1 96

Table 2.7. Effect of various carbohydrates on hydrogen evolution and evolution rate by Rhodopseudomonas sphaeroides K-7 97

Table 2.8. Effect of amino acids on hydrogen evolution by Rhodopseudomonas sp. E 15-1 99

Table 2.9. Effect of amino acids on hydrogen evolution and evolution rate by Rhodopseudomonas sphaeroides K-7 100

Table 2.10. Effect of various carbohydrates on hydrogen evolution by mixed culture of Cl.butyricum NCIB 9576 and R.sphaeroides K-7 101

Table 2.11. Effect of various amino acids on hydrogen evolution by mixed culture of Cl.butyricum NCIB 9576 and R.sphaeroides K-7 103

Table 2.12. Effect of amino acids on hydrogen evolution by mixed culture of Cl.butyricum NCIB 9576 and R.sphaeroides K-7 104

Table 2.13. Effect of various carbohydrates on hydrogen evolution which were added to the waste water medium by mixed culture of Cl.butyricum NCIB 9576 and R.sphaeroides K-7 128

Table 2.14. Effect of various organic acids on hydrogen evolution which were added to the waste water medium by mixed culture of Cl.butyricum NCIB 9576 and R.sphaeroides K-7 130

Fig.1.1. Experimental procedure of medium preparation, incubation and assay of fermentation products in batch culture 23

Fig.1.2. Schematic diagram of hydrogen evolution in semi-continuous culture system 1. Argon gas tank 2. Reactor vessel 3. Culture medium 4. Magnetic stirrer 5. Fresh medium reservoir 6. Peristaltic pump 7. Spent medium reservoir 8. Produced gas... 25

Fig.1.3. Fermentation system used in semi-continuous culture 26

Fig.1.4. Experimental procedure of semi-continuous culture and assay of fermentation products 28

Fig.1.5. Fermentation products of Bacillus sp. I-17 in modified PYG medium containing glucose of 1% in batch culture Incubation temperature : 30℃ 33

Fig.1.6. Fermentation products of Bacillus sp. I-19 in modified PYG medium containing xylose of 1% in batch culture Incubation temperature : 37℃ 34

Fig.1.7. Growth curves of Bacillus sp. I-17 and I-19 ●-● : Cell mass of Bacillus sp. I-17 at 30℃ ○-○ : Cell mass of Bacillus sp. I-19 at 37℃(이미지참조) 36

Fig.1.8. Specific growth rate of Bacillus sp. I-17 at 30℃ ● : Cell mass of Bacillus sp. I-17 at 30℃ in log scale μ₁ : Specific growth rate of Bacillus sp. I-17 at 30 ℃ 37

Fig.1.9. Specific growth rate of Bacillus sp. I-19 at 37℃ ○ : Cell mass of Bacillus sp. I-19 at 37℃ in log scale μ₂ : Specific growth rate of Bacillus sp. I-19 at 37℃ 38

Fig.1.10. Hydrogen evolution of Bacillus sp. I-17 in semi-continuous feeding Spent medium replaced with fresh medium containing glucose of 1% at 50% replacement rate Semi-continuous culture performed at 30℃ ↓(이미지참조) indicate the time of... 39

Fig.1.11. Hydrogen evolution of Bacillus sp. I-17 in semi-continuous feeding Spent medium replaced with fresh medium containing glucose of 1% at 40% replacement rate Semi-continuous culture performed at 30℃ ↓(이미지참조) indicate the time of... 40

Fig.1.12. Hydrogen evolution of Bacillus sp. I-17 in semi-continuous feeding Spent medium replaced with fresh medium containing glucose of 1% at 30% replacement rate Semi-continuous culture performed at 30℃ ↓(이미지참조) indicate the time of... 41

Fig.1.13. Hydrogen evolution of Bacillus sp. I-17 in semi-continuous feeding Spent medium replaced with fresh medium containing glucose of 1% at 20% replacement rate Semi-continuous culture performed at 30℃ ↓(이미지참조) indicate the time of... 42

Fig.1.14. Hydrogen evolution of Bacillus sp. I-19 in semi-continuous feeding Spent medium replaced with fresh medium containing xylose of 1% at 50% replacement rate Semi-continuous culture performed at 37℃ ↓(이미지참조) indicate the time of... 43

Fig.1.15. Hydrogen evolution of Bacillus sp. I-19 in semi-continuous feeding Spent medium replaced with fresh medium containing xylose of 1% at 40% replacement rate Semi-continuous culture performed at 37℃ ↓(이미지참조) indicate the time of... 44

Fig.1.16. Hydrogen evolution of Bacillus sp. I-19 in semi-continuous feeding Spent medium replaced with fresh medium containing xylose of 1% at 30% replacement rate Semi-continuous culture performed at 37℃ ↓(이미지참조) indicate the time of... 45

Fig.1.17. Hydrogen evolution of Bacillus sp. I-19 in semi-continuous feeding Spent medium replaced with fresh medium containing xylose of 1% at 20% replacement rate Semi-continuous culture performed at 37℃ ↓(이미지참조) indicate the time of... 46

Fig.1.18. Effect of the glucose concentration on hydrogen evolution of Bacillus sp. I-17 at 50% replacement rate in semi-continuous culture Incubation temperature : 37℃ Semi-continuous feeding started after 12hrs of batchwise culture Time... 50

Fig.1.19. Effect of the glucose concentration on hydrogen evolution of Bacillus sp. I-17 at 30% replacement rate in semi-continuous culture Incubation temperature : 37℃ Semi-continuous feeding started after 13hrs of batchwise culture Time... 51

Fig.1.20. Effect of the glucose concentration on hydrogen evolution of Bacillus sp. I-17 at 20% replacement rate in semi-continuous culture Incubation temperature : 37℃ Semi-continuous feeding started after 11hrs of batchwise culture Time... 52

Fig.1.21. Effect of the temperature on hydrogen evolution of Bacillus sp. I-17 Modified PYG media containing glucose of 1% was used ●-●: Hydrogen evolution at 30℃ ●-●: Hydrogen evolution at 37℃ ▲-▲: Cell growth at 30℃ ■-■:...(이미지참조) 54

Fig.1.22. Effect of the agitation speed on hydrogen evolution of Bacillus sp. I-17 in semi-continuous culture Incubation temperature : 37℃ Semi-continuous feeding started after 12hrs of batchwise culture 100ml of spent medium was replaced with... 56

Fig.1.23. Effect of the inoculum level on hydrogen evolution and cell growth of Bacillus sp. I-17 Incubation temperature : 37℃ PYG media containing glucose of 1% and products assay was performed after 12hrs and 24hrs ●-●: H₂...(이미지참조) 57

Fig.1.24. Effect of cysteine HCI and vitamins (Biotin, p-Aminobenzoic acid) on hydrogen evolution and cell growth of Bacillus sp. I-17 ●-●: Hydrogen evolution, ●- -●:Cell growth at (-)Y (-)P (+)C (+)V ○-○: Hydrogen evolution,...(이미지참조) 61

Fig.1.25. Cell growth of Bacillus sp. I-17 at various concentration of yeast extract and peptone, and inorganic components in semi-continuous culture (A) ● : 75% (B) ▲ : 50% (C) ■ : 25%(이미지참조) 64

Fig.1.26. Effect of the glutamate concentration on hydrogen evolution of Bacillus sp. I-17 in semi-continuous culture 66

Fig.2.1. Branched catabolic pathway of glucose fermentation by Clostridium butyricum 79

Fig.2.2. Experimental procedure of medium preparation, incubation and assay of fermentation products in batch culture 86

Fig.2.3. Hydrogen evolution and fermentation product by mixed culture of Cl.butyricum NCIB 9576 and R.sphaeroides K-7 when glucose was added to Ormerod medium as a carbon source. 105

Fig.2.4. Hydrogen evolution and fermentation product by mixed culture of Cl.butyricum NCIB 9576 and R.sphaeroides K-7 when lactose was added to Ormerod medium as a carbon source. 106

Fig.2.5. Hydrogen evolution and fermentation product by mixed culture of Cl.butyricum NCIB 9576 and R.sphaeroides K-7 when sucrose was added to Ormerod medium as a carbon source. 107

Fig.2.6. Effect of the starch concentration on hydrogen evolution by mixed culture of Cl.butyricum NCIB 9576 and R.sphaeroides K-7 ( ○ : 0.5% starch, ● : 1% starch, ▲ : 2% starch, △ : 5% starch, ■ : 10% starch)(이미지참조) 109

Fig.2.7. Hydrogen evolution and cell growth by mixed culture of Cl.butyricum NCIB 9576 and R.sphaeroides K-7 when added starch was 0.5% 110

Fig.2.8. Hydrogen evolution and cell growth by mixed culture of Cl.butyricum NCIB 9576 and R.sphaeroides K-7 when added starch was 1% 111

Fig.2.9. Hydrogen evolution and cell growth by mixed culture of Cl.butyricum NCIB 9576 and R.sphaeroides K-7 when added starch was 2% 112

Fig.2.10. Hydrogen evolution and cell growth by mixed culture of Cl.butyricum NCIB 9576 and R.sphaeroides K-7 when added starch was 5% 113

Fig.2.11. Hydrogen evolution and cell growth by mixed culture of Cl.butyricum NCIB 9576 and R.sphaeroides K-7 when added starch was 10% 114

Fig.2.12. Effect of the L-glutamate concentration on hydrogen evolution and cell growth of Bacillus sp. I-17. Hydrogen evolution and biomass were determined after 48 hr. incubation, ( ● : Hydrogen evolution, ○ : Biomass)(이미지참조) 116

Fig.2.13. Effect of the L-glutamate concentration on hydrogen evolution and cell growth of Bacillus sp. I-19. Hydrogen evolution and biomass were determined after 48 hr. incubation. ( ● : Hydrogen evolution, ○ : Biomass)(이미지참조) 117

Fig.2.14. Effect of the L-glutamate concentration on hydrogen evolution and cell growth of Cl.butyricum NCIB 9576. Hydrogen evolution and biomass were determined after 96 hr. incubation. ( ●:Hydrogen evolution, ○:biomass when 0% yeast extract and peptone. ▲:Hydrogen evolution, △:biomass when 50% yeast extract and peptone. ■:Hydrogen... 118

Fig.2.15. Effect of the L-Glutamate concentration on hydrogen evolution and cell growth of R.acidophila A 6-4. Hydrogen evolution and biomass were determined after 168 hr. incubation. ( ● : Hydrogen evolution, ○ : Biomass)(이미지참조) 119

Fig.2.16. Effect of the L-Glutamate concentration on hydrogen evolution and cell growth of R.acidophila B 1-2. Hydrogen evolution and biomass were determined after 168 hr. incubation. ( ● : Hydrogen evolution, ○ : Biomass)(이미지참조) 120

Fig.2.17. Effect of the L-Glutamate concentration on hydrogen evolution and cell growth of Rhodopseudomonas sp. E 15-1. Hydrogen evolution and biomass were determined after 168 hr. incubation. (● : Hydrogen evolution, ○ : Biomass)(이미지참조) 121

Fig.2.18. Effect of the L-Glutamate concentration on hydrogen evolution and cell growth of R.sphaeroides K-7. Hydrogen evolution and biomass were determined after 168 hr. incubation. ( ● : Hydrogen evolution, ○ : Biomass)(이미지참조) 122

Fig.2.19. Effect of the L-Glutamate concentration on hydrogen evolution and cell growth by mixed culture of Cl.butyricum NCIB 9576 and R.sphaeroides K-7. Hydrogen evolution and biomass were determined after 168 hr. incubation. ( ● : Hydrogen evolution, ○ : Biomass)(이미지참조) 123

Fig.2.20. Effect of the organic waste water concentration on hydrogen evolution of Bacillus sp. and Cl.butyricum NCIB 9576. ( ● : Bacillus sp. I-17, ▲ : Bacillus sp. I-19, ■ : CI. butyricum NCIB 9576) 125

Fig.2.21. Effect of the organic waste water concentration on hydrogen evolution of Rhodopseudomonas sp. and mixed culture. 127

Fig.2.22. Hydrogen evolution and waste water treatment by mixed culture of Cl.butyricum NCIB 9576 and R.sphaeroides K-7. when 1% starch was added to waste water medium 132

Fig.2.23. Hydrogen evolution and waste water treatment by mixed culture of Cl.butyricum NCIB 9576 and R.sphaeroides K-7 when 30mM lactose was added to waste water medium 133

Fig.2.24. Hydrogen evolution and waste water treatment by mixed culture of Cl.butyricum NCIB 9576 and R.sphaeroides K-7 when 30mM maltose was added to waste water medium. 134