In situ 측정기법에 의한 호기성 고정생물막내 질산화균의 활성응답 특성 = Characteristics of activity response of nitrifying bacteria in aerobic fixed-biofilm through in situ analysis methods

다국어 초록 (Multilingual Abstract)

The activity response and spatial distribution of nitrifying bacteria in an aerobic fixed-biofilm were analyzed by in situ analysis techniques, such as SEM (scanning electron microscopy), CSLM (confocal scanning laser microscopy), ATP bioluminescence, FISH (fluorescence in situ hybridization) and microelectrodes depending on dilution rates. This study show the characteristics of a fixed-biofilm attachment and growth through optical approach with SEM, image camera and CSLM, the activity response of nitrifying bacteria and biofilm with ATP bioluminescence technique, monitoring the spatial distribution and activity of nitrifying bacteria with combined FISH with microelectrods system. Aerobic fixed-biofilms were grown in four bench-scale fixed-bed reactors (6.7L) that were packed with 20% (v/v) cubic acryl-media. The dilution rates were 0.10, 0.13, 0.17 and 0.25h^(-1), respectively.
Through this study, conclusions were derived as fellow;
Characteristics of attachment and improvement of an aerobic fixed-biofilm by optical approach.
The aerobic fixed-biofilm had heterogeneous and non-uniformed structure that contained amount of cell cluster, void and channel with SEM, image camera and CSLM. Biofilm formation occurred attachment to a surface, growth of microbes on the surface, and detachment of microbes from surfaces, simultaneously. The initial colonization of a clean surface, although restricted to a subset of the whole population that can colonize surfaces, should be entirely random. Biofilm grew to 200㎛ thickness of 2 day and 1,200㎛ thickness of 20 day. On day 32, detachment of microbes with low adhesion occurred at the mature biofilm due to respiration and cell lysis within biofilm. The depth profiles of areal porosity are directly related to biofilm porosity. Biofilm porosity of aerobic fixed-biofilm reconstructed by CSLM was 0.84 on 25-day-biofilm at dilution rate of 0.10h^(-1).
Response of dynamic activity for nitrifying bacteria with variation of dilution rates in an aerobic fixed-biofilm using ATP bioluminescence technique.
The existing analysis methods for measurement of biofilm activity had a shortcoming to be spend from a few hours to several days. In order to overcome the shortcoming, ATP monitoring technique using ATP monitoring kit, required more relatively convenience and shorter analysis time to measure the biofilm activity, was applied. The light intensity of RLUs (relative light units) was increased, while the amount of active nitrifying bacteria was increased in the biofilm. Biomass quality (measured by SATP) and biomass activity (measured by INT-DHA activity) are closely correlated in an aerobic fixed-biofilm (R^(2)=0.94). When the removal rate of NH_(4)^(+)-N was ranged in between 88.1∼224.8g NH_(4)^(+)-N/㎥·d, specific ATP (SATP) was measured in 0.10∼0.35 mg ATP/g DS. INT-DHA activity increased from 71.4 to 96.2 mg O_(2)/g VSS·d as over the same increase in dilution rate. The SATP of wastewater biofilm and autotrophs biofilm was increased from 0.11 to 0.24 mg ATP/g DS and from 0.08 to 0.17 mg ATP/g DS respectively. However, the SATP of nitrifying bacteria of wastewater biofilm was ranged 0.04∼0.08 mg ATP/g DS. Results from the aerobic fixed-biofilm showed ATP concentration for nitrifying bacteria was 2×10^(-9)㎍ ATP/cell. qSATP estimated by ATP simulation equation was 0.32∼0.60. Consequently, ATP bioluminescence technique has made it is possible to analyze the activity of fixed-biofilm and to provide a means of rapidly and accurately quantitative analysis as the monitoring tool.
Characteristics of growth activity and distribution of nitrifying bacteia in an aerobic fixed-biofilm through fluorescence in situ hybridization (FISH).
To investigate spatial distribution of nitrifying bacteria and variation of microenvironment in aerobic fixed-biofilms, fluorescent in situ hybridization (FISH) method was used depending on dilution rates. The specific cell numbers and specific cell area of each population were determined by visual digital image analyser. When dilution rates was increased from 0.10 to 0.25h^(-1), total cell number by EUB338 and DAPI staining was decreased from 5.24×10^(12) to 1.41×10^(12) cell/㎤. The microbial compositions, using EUB338, BET42a and Nsm156 probe, were β-proteobacteria (ammonia-oxidizing bacteria: AOB) of 13.5∼8.2% and Nitrosomonas spp. of 11.8∼5.8%, respectively according to increasing from 0.10 to 0.25h^(-1) of dilution rate. On day 25, the distribution of AOB with depth of biofilm was 13.3% of 400㎛ at dilution rate of 0.10h^(-1) and was 13.9% of 200㎛ at dilution rate of 0.25h^(-1). Using FISH with EUB338 and BET42a on 35-day-biofilm, the distribution area of AOB at dilution rate of 0.10h^(-1) was higher 11% than that of 0.17h^(-1). Using Beta42 and Nb1000, Nitrobacter spp. at dilution rate of 0.17h^(-1) was higher 5% than that of 0.10h^(-1). If the cell number was equal to microbial count detected by EUB338, the distribution of Nitrosomonas and Nitrobacter by INT method was 0.15∼0.22 and 0.12∼0.17, respectively according to decreasing from 0.25 to 0.10h^(-1). Using FISH with vertical section to monitor spatial distribution of AOB, they extended their area of growth to the outer part of the biofilm previously occupied by heterotrophs and low numbers of nitrifying bacteria were also participated in the bottom of the biofilm. The decrease in organic compounds limited the growth of heterotrophs, and nitrifying bacteria could grow in the outer part of the biofilm with the low C/N ratio. In contrast, the dominance of nitrifying bacteria in the aerobic biofilm with the high concentration of organic compounds and the low concentration of bulk liquid O_(2) was formed deeper inside the biofilm due to be poor competitiveness with heterotrophs. FISH with rRNA probes has made it possible to analyze complex in situ microbial community structures and to diagnosis biofilm reactors. FISH also demonstrated to determine the composition of microbial population as a powerful tool in biofilms.
Measurement of substrate concentration profile and calculation of effective coefficient in an aerobic fixed-biofilm using microelectrodes
Response time of fabricated ISEs was short within 1min and had over 5% of standard error, but the correlation(R^(2)) was 0.98. Using ISEs on 35-day-biofilm, NH_(4)^(+)-N concentration profile was decreased with O_(2) profile up to 800 ㎛ depth of biofilm. On the contrary, NO_(3)^(￣)-N profile was increased until the depth of 800 ㎛ and was slightly decreased the depth after. Using O_(2) microelectrode, oxygen concentration was constant at the bulk phase, but the slope was sharply decreased from biofilm surface to substratum according to growing up biofilm. As biofilm thickness was thicker, the biofilm density would be high and aerobic and anoxic zone have coexisted at near substratum. As the dilution rate increased from 0.10 to 0.25h^(-1), slop of DO concentration profile within biofilm was increased and the depth of oxygen transfer was decreased from 1,500 ㎛ to 1,000 ㎛ of biofilm thickness. As the dilution rate was increased, D_(s) was also increased. Therefore, it explained that the depth of oxygen transfer was declined because oxygen uptake rate per bacteria and biofilm density were increased by increased substrate loading rate and velocity according to increased from 0.10 to the dilution rate of 0.25h^(-1).
Finally, in situ analysis techniques (ATP bioluminescence, FISH and microelect-rode) made it is possible to diagnosis and estimate the phenomena of nitrification inhibition was not well explained with microbial population but microbial activity. It could be need the integrated in situ analysis approach to analyze the performance of nitrification and reactor in biofilm systems.

목차 (Table of Contents)

• 목차 =ⅰ
• LIST OF FIGURES = ⅳ
• LIST OF TABLES = ⅹ
• NOMENCLATURE = xi
• 제1장 서론 = 1
• 1.1 연구배경 및 목적 = 1
• 1.2 논문 구성 = 5
• 제2장 문헌연구 = 6
• 2.1 질산화(Nitrification) = 6
• 2.2 생물막 형성 및 구조적 특성 = 17
• 2.3 생물막 메커니즘 연구 = 40
• 2.3.1 미소전극(Microelectrode) = 40
• 2.3.2 Fluorescence In Situ hybridization(FISH) = 49
• 2.4 생물막내 물질전달 모델 = 64
• 제3장 형태학적 분석을 통한 호기성 고정생물막의 부착 및 성장과 물리적 변화 특성 = 71
• 3.1 서론 = 71
• 3.2 실험 재료 및 방법 = 73
• 3.2.1 실험장치 = 73
• 3.2.2 운전조건 및 하수특성 = 73
• 3.2.3 분석방법 = 76
• 3.3 결과 및 고찰 = 77
• 3.3.1 형태학적 접근에 의한 부착 생물막 관찰 = 78
• 3.3.3 생물막 구조적 특성 = 84
• 3.3.4 생물막 두께에 따른 밀도 변화 = 88
• 3.4 중간 결론 = 92
• 제4장 ATP bioluminscence를 활용한 호기성 생물막내 희석률에 따른 질산화 미생물의 응답 특성 = 93
• 4.1 서론 = 93
• 4.2 실험 장치 및 방법 = 95
• 4.2.1 실험장치 = 95
• 4.2.2 운전조건 및 하수특성 = 95
• 4.2.2 분석방법 = 97
• 4.3 결과 및 고찰 = 102
• 4.3.1 희석률에 따른 질소제거 = 102
• 4.3.2 Bioluminescence 반응에 대한 ATP 생성반응 = 104
• 4.3.3 호기성 생물막 활성도 평가 = 106
• 4.3.4 호기성 생물막의 ATP 시뮬레이션 평가 = 113
• 4.4 중간 결론 = 118
• 제5장 In Situ Hybridization에 의한 호기성 생물막내 질산화균의 성장활성 및 분포특성 = 120
• 5.1 서론 = 120
• 5.2 실험 재료 및 방법 = 122
• 5.2.1 실험장치 = 122
• 5.2.2 운전조건 및 하수특성 = 122
• 5.2.3 분석방법 = 124
• 5.3 결과 및 고찰 = 129
• 5.3.1 In situ hybridization에 의한 질산화균의 공간분포 특성 = 129
• 5.3.2 INT 법에 의한 미생물종의 분포 특성 = 148
• 5.3.3 희석률에 따른 생물막내 종 분포 변화 = 150
• 5.4 중간 결론 = 153
• 제6장 미소전극을 이용한 호기성 고정생물막내 기질농도 변화와 산소 유효확산 계수 결정 = 155
• 6.1 서론 = 155
• 6.2 실험 장치 및 방법 = 157
• 6.2.1 실험장치 및 운전 = 157
• 6.2.2 운전조건 및 하수특성 = 157
• 6.2.3 실험방법 = 158
• 6.3 산소미소전극에 의한 생물막내 동력학 변수결정 = 162
• 6.4 결과 및 고찰 = 164
• 6.4.1 ISE 미소전극을 이용한 생물막내 이온성 기질농도 분포 변화 = 165
• 6.4.2 생물막내 산소농도 분포 및 미생물 형태 = 174
• 6.4.3 산소전극을 이용한 생물막내 반포화 속도 상수 및 유효확산 계수 결정 = 180
• 6.4 중간 결론 = 189
• 제7장 결론 = 191
• 참고문헌 = 195
• Abstract = 212