표제지

제출문

보고서 초록

요약문

SUMMARY

Contents

목차

제1장 연구개발과제의 개요 32

제1절 연구개발의 필요성 및 목적 34

제2절 연구개발의 내용 및 범위 35

제2장 국내외 기술개발 현황 38

제1절 국내의 기술개발 현황 40

제2절 국외의 기술개발 현황 42

1. 단축질소제거 메카니즘 42

2. Anammox의 대사 메커니즘 46

3. Anammox 공정의 적용 50

4. Anammox 공정내 미생물 군집 분포 52

가. Fluorescence in-situ hybridization (FISH) 54

나. Design of group- specific oligonucleotide probes 57

다. rDNA library and sequencing 58

제3장 연구개발수행 내용 및 결과 64

제1절 연구개발 수행 내용 66

1. 연속배양을 통한 Anammox균의 고농도배양조건 확립 66

2. Anammox 입상 슬러지 생산 및 안정성 평가 66

3. 초고율 생물반응기의 장기운전 및 성능평가 67

제2절 연구결과 및 고찰 68

1. 초고율 질소 제거 시스템 구성을 위한 안정적 배양 기술 확보 68

가. 개요 68

나. 연속 반응기의 특징 및 분석 방법 68

(1) 연속반응기의 특징 68

(2) 유입 합성폐수의 특성 72

(3) 반응가스 측정 및 화학분석 73

다. 연속운전 결과 74

(1) 부직포를 충진한 연속 배양기(R1)의 운전 74

(2) 부직포 배양기와 UASB를 결합한 연속 배양기(R2)의 운전 83

(3) 탄소섬유 반응기와 UASB를 결합한 연속 배양기(R3)의 운전 93

(4) 질소 제거 시스템의 Anammox 반응 비교ㆍ평가 102

(가) 본 연구에 사용된 연속 배양기의 성능 비교 102

(나) NO₂-N가 Anammox 반응에 미치는 영향 분석 103

라. 실험결과 요약 108

2. 분자생물학적 기법을 이용한 미생물의 규명 및 정량 109

가. Fluorescence in situ hybridization (FISH) 109

(1) Cell fixation 및 granule 미세절단 109

(2) in situ hybridization 방법 109

(3) Fluorescence in situ hybridization (FISH) 결과 110

나. DNA 정보의 계통분류학적 분석 122

(1) DNA 추출 및 PCR amplication 122

(2) Gene Cloning 및 Transformation 122

(3) DNA 정보의 계통분류학적 분석결과 123

다. Real-time quatitative PCR을 이용한 anammox 미생물의 정량 133

(1) 개요 133

(2) Real-time qPCR 133

(3) Real-time qPCR 활용 가능성 타진 134

(4) primer set 선정 135

라. T-RFLP를 이용한 미생물 군집구조 분석 138

(1) 개요 138

(2) T-RFLP 138

(3) 실험목적 139

(4) 실험방법 139

(5) T-RFLP 실험결과 140

(가) 미생물 식별을 위한 데이터베이스 구축 140

(나) anammox 미생물 T-RF 식별 실험 142

(다) 미생물 군집구조 변화 분석 142

(6) 결론 144

마. 결과요약 145

3. Anammox 미생물의 입상화 및 적용성 평가 146

가. 개요 146

나. 실험방법 146

(1) 생물학적 및 화학적 입상화 146

(2) 고율 혐기성 암모늄 산화 반응기의 설계 146

(3) 고율 혐기성 암모늄 산화반응기의 특징 146

(4) 합성폐수의 특성 148

(5) 반응가스 측정 및 화학분석 148

다. 실험결과 150

(1) 생물학적 입상화 150

(2) 화학적 입상화 150

(3) 입상슬러지의 적용성 평가 152

라. 결과요약 154

4. 고율 혐기성 암모늄 산화균의 실폐수 적용성 평가 155

가. 개요 155

나. 실험장치 및 운전조건 155

(1) 실험장치 155

(2) 반류수의 특징 155

다. 실험결과 156

라. 결과요약 157

제3절 총괄 결론 158

제4장 목표 달성도 및 관련분야에의 기여도 162

제1절 목표달성도 164

1. 기술개발 추진체계 164

가. 최대 질소제거 부하율 165

나. Anammox 균주의 우점화 정도 165

다. 입상 Anammox 미생물의 활성도 165

2. 최종목표 166

제2절 관련분야에의 기여도 168

1. 관련분야의 기술발전의 기여도 168

2. 관련분야의 기술발전의 활용실적 170

가. 국내외 논문게재 170

나. 국내외 학술회의 발표 171

다. 특허등록 및 출원 172

제5장 개발결과의 활용계획 174

제6장 연구개발과정에서 수집한 해외과학기술정보 178

제7장 참고문헌 184

Table 2-1. Effluent standards of sewage treatment plants in Korea. 40

Table 2-2. Evaluation of practical water quality compared with goals 41

Table 2-3. Comparison of operating parameters in biological nitrogen removal process. 50

Table 2-4. Applications of anammox bacteria. 51

Table 3-1. Composition of seeding sludge in continuous culture reactors. 71

Table 3-2. Composition of synthetic medium used this study. 72

Table 3-3. Comparisons of reactor performances. 102

Table 3-4. Comparison of anammox reaction molar ratio. 103

Table 3-5. Highest level of nitrite nitrogen applied to anammox reaction. 105

Table 3-6. rRNA targeted oligonucleotide probes used this study. 110

Table 3-7. PCR primers used in this study. 122

Table 3-8. Alignment of target sequences of the Pla46 probe suite with the corresponding region of 16S rRNA. Mismatches with the Pla46 probe sequence are shaded. 131

Table 3-9. Alignment of target sequences of the Amx368 probe suite with the corresponding region of 16S rRNA. Mismatches with the Amx368 probe sequence are shaded. 132

Table 3-10. T-RF Database for analysis of temporal diversity of microbial community structure of anammox process. 141

Table 3-11. Identified microorganisms during disturbance by using T-RFLP. 143

Table 3-12. Composition of synthetic medium used this study. 149

Table 3-13. Operational results of hybrid reactor. 153

Table 3-14. The characteristics of recycle water used this study. 156

Table 3-15. Operational results of recycle wastewater treatment. 157

Table 4-1. Evaluation method and items of this study. 166

Table 4-2. Evaluation method and items of this study during this year. 167

Table 4-3. Cost-benefit analysis of anammox process. 169

Table 6-1. The start-up time of anammox reactor decreased significantly with gained experience and availability of seed sludge. 181

Figure 2-1. Microbial nitrogen cycle. 43

Figure 2-2. Minimum residence time for ammonium and nitrite oxidisers as function of the temperature (Hellinga et al., 1998). 44

Figure 2-3. Reduction of oxygen and C-source requirements by N-removal via nitrite. 45

Figure 2-4. Possible pathway for Anaerobic ammonium oxidation(ANAMMOX) by the Planctomycetes. 47

Figure 2-5. Possible reaction mechanisms and cellular localization of the enzyme systems involved in anaerobic ammonium oxidation. 47

Figure 2-6. A; Three-dimensional visualisation of the staircase structure of the 5-cyclobutane ladderane moiety. B:Structures of various ladderane lipids of " Candidatus B. anammoxidans" 48

Figure 2-7. Commonly approaches using nucleic acids in molecular microbial ecology. 53

Figure 2-8. Diversity of deep-branching planctomycetes. 54

Figure 2-9. Principles of in situ hybridization. 56

Figure 2-10. The procedure of 16S rDNA library. 58

Figure 2-11. Principle of cloning 59

Figure 2-12. Map and Sequence reference points of the T&A cloning vector 60

Figure 2-13. Principle of Ampicillin selection & LacZ color selection. 61

Figure 3-1. Schematic diagram and a photos of continuous culture reactors 69

Figure 3-2. Schematic diagram of the R1 and R2 reactors. 70

Figure 3-3. Schematic diagram of the R3 reactor. 71

Figure 3-4. Variation of ammonia nitrogen concentration in R1 reactor 75

Figure 3-5. Variation of nitrite nitrogen concentration in R1 reactor. 75

Figure 3-6. Variation of ammonium nitrogen removal efficiency with nitrogen loading rate applied in R1 reactor. 77

Figure 3-7. Variation of nitrite nitrogen removal efficiency with nitrogen loading rate applied in R1 reactor. 77

Figure 3-8. Variation of produced nitrate nitrogen concentration in R1 reactor 78

Figure 3-9. Nitrogen loading rate applied vs nitrogen loading rate removed. 78

Figure 3-10. Variation of T-N concentration and T-N removal efficiency in R1 reactor 80

Figure 3-11. Variation of nitrogen gas production in R1 reactor. 80

Figure 3-12. Variation of ORP in R1 reactor. 81

Figure 3-13. Variation of pH in R1 reactor. 81

Figure 3-14. Variation of alkalinity in R1 reactor. 82

Figure 3-15. Variation of removed alkalinity in R1 reactor. 82

Figure 3-16. Variation of ammonium nitrogen concentration in R2 reactor. 84

Figure 3-17. Variation of nitrite nitrogen concentration in R2 reactor. 85

Figure 3-18. Variation of ammonium nitrogen removal efficiency with nitrogen loading applied in R2 reactor. 86

Figure 3-19. Variation of nitrite nitrogen removal efficiency with nitrogen loading applied in R2 reactor. 87

Figure 3-20. Variation of produced nitrate concentration in the R2 reactor. 87

Figure 3-21. Nitrogen loading rate applied vs nitrogen loading rate removed. 88

Figure 3-22. Variation of T-N concentration and T-N removal efficiency in R2 reactor 90

Figure 3-23. Variation of the daily nitrogen gas production in R2 reactor. 90

Figure 3-24. Variation of ORP in R2 reactor. 91

Figure 3-25. Variation of pH in R2 reactor. 91

Figure 3-26. Variation of alkalinity in R2 reactor. 92

Figure 3-27. Variation of removed alkalinity in R2 reactor. 92

Figure 3-28. Variation of ammonia nitrogen concentration in R3 reactor. 94

Figure 3-29. Variation of nitrite nitrogen concentration in R3 reactor. 95

Figure 3-30. Variation of free ammonia concentration in R3 reactor. 95

Figure 3-31. Variation of ammonium removal efficiency with nitrogen loading rate applied in R3 reactor. 96

Figure 3-32. Variation of nitrite removal efficiency with nitrogen loading rate applied in R3 reactor. 96

Figure 3-33. Variation of produced nitrate concentration in R3 reactor. 98

Figure 3-34. Nitrogen loading rate applied vs nitrogen loading rate removed 98

Figure 3-35. Variation of the daily nitrogen production in R3 reactor 99

Figure 3-36. Variation of ORP in R3 reactor. 99

Figure 3-37. Variation of pH in R3 reactor. 100

Figure 3-38. Variation of alkalinity in R3 reactor. 100

Figure 3-39. Variation of removed alkalinity in R3 reactor. 101

Figure 3-40. Ammonium nitrogen removal efficiency according to nitrite nitrogen concentration in R1. 104

Figure 3-41. Ammonium nitrogen removal efficiency according to nitrite nitrogen concentration in R2. 104

Figure 3-42. Ammonium nitrogen removal efficiency according to nitrite nitrogen concentration in R3. 105

Figure 3-43. Relationship between free ammonia concentration and ammonium nitrogen removal efficiency in R1. 106

Figure 3-44. Relationship between free ammonia concentration and total nitrogen removal efficiency in R3. 107

Figure 3-45. Change of the granule 111

Figure 3-46. Fluorescence in situ hybridization of granules in R-2 continuous reactor. 112

Figure 3-47. Fluorescence in situ hybridization of granules in R-2 continuous reactor. 113

Figure 3-48. Fluorescence in situ hybridization of granules in R-2 continuous reactor. 114

Figure 3-49. Fluorescence in situ hybridization of granules in R-2 continuous reactor. 115

Figure 3-50. Fluorescence in situ hybridization of granules in R-2 continuous reactor. 115

Figure 3-51. Fluorescence in situ hybridization of granules in R3 reactor red granule. 116

Figure 3-52. Fluorescence in situ hybridization of granules in R3 continuous reactor. 117

Figure 3-53. Fluorescence in situ hybridization of granules in R3 reactor red granule. 118

Figure 3-54. Fluorescence in situ hybridization of granules in R3 reactor red granule. 119

Figure 3-55. Fluorescence in situ hybridization of granules in R3 reactor red granule. 119

Figure 3-56. Fluorescence in situ hybridization of granules in R-3 continuous reactor. 120

Figure 3-57. PCR amplication of 16S rRNA gene fragment amplified with EUB 27F and EUB 1492R primers. 124

Figure 3-58. Hind III insert digestion based on 16S rRNA gene fragment amplified. 124

Figure 3-59. Phylogenetic tree of mixed granule in the R2 UASB reactor(Partial 16S rRNA gene sequence). 125

Figure 3-60. Phylogenetic tree of red granule in the R3 UASB reactor(16S rRNA gene Full sequence). 127

Figure 3-61. Phylogenetic tree of mixed granule in the R3 UASB reactor(16S rRNA gene Full sequence). 129

Figure 3-62. Phylogenetic tree of sludge in the R3 UASB reactor(16S rRNA gene Full sequence). 130

Figure 3-63. Mechanisms of PCR and fluorescence quantification. 135

Figure 3-64. PCR product of primer set of PLA46 and mod-AMX368. 136

Figure 3-65. Expectation of secondary structure of PLA46 and AMX36. 137

Figure 3-66. Production of primr dimer of PLA46 and d-mod-AMX368. 137

Figure 3-67. Procedure of T-RFLP. 139

Figure 3-68. Sampling points during disturbance of R3 reactor. 140

Figure 3-69. Identification of positive T-RF of anammox clone. 142

Figure 3-70. Analysis of microbial community structure using T-RFLP. 143

Figure 3-71. Comparison of microbial community structure in time series using T-RFLP results. 144

Figure 3-72. Schematic diagram of the hybrid anammox reactor. 147

Figure 3-73. Change of the granule. 150

Figure 3-74. Determination of dose of cationic polymer. 151

Figure 3-75. Size distribution of the chemical anammox granule. 151

Figure 3-76. Comparison of settling velocity according to mixing ratio between anaerobic digested sludge and anammox sludge 152

Figure 4-1. Representative characteristics of reject water in large plants. 168

Figure 4-2. Representative characteristics of reject water in smaller plants. 169