정족수감지 억제 미생물 고정화 판형담체 개발과 셀룰로오스 분해 미생물의 적용을 통한 하폐수처리용 분리막 생물반응기에서의 생물막오염 제어

학위논문 (박사)-- 서울대학교 대학원 : 화학생물공학부, 2017. 2. 이정학.최근, 분리막 생물반응기(Membrane Bioreactor, MBR) 내 고질적인 문제인 생물막오염(Biofouling)을 근본적으로 해결하고자 미생물 간의 대화 (Quorum sensing, QS)를 차단하는 정족수감지 억제 (Quorum quenching, QQ) 기술을 적용한 사례가 활발히 보고되고 있다. 하지만 지금까지 개발된 QQ 기술 (즉, QQ 미생물 담체 개발)은 평막 또는 실제 분리막모듈과 거리가 먼 일자형의 중공사모듈에서만 적용해왔기 때문에 다발형의 중공사모듈에서 QQ 기술의 생물막오염 제어 성능 확인이 필요하다. 또한, QQ 기술은 생물막이 형성된 이후에는 제어에 효과적이지 않기 때문에 이미 형성된 생물막를 제어할 수 있는 방안도 필요하다. 따라서, 본 연구에서는 분리막 시장에서 가장 많이 사용되고 있는 다발형의 중공사모듈에서 생물막오염 억제가능성을 확인하기 위해 새로운 형태의 QQ 미생물 담체를 개발하였다. 또한, 이미 형성된 생물막을 제어하기 위한 방안으로 생물막 내에 존재하는 셀룰로오스를 분해할 수 있는 미생물을 분리, 동정하고 담체에 고정시켜 MBR에 적용하여 또 다른 생물막오염 제어 방법의 가능성을 확인하였다. 첫째, 새로운 모양인 QQ 미생물 판형담체 (QQ-sheets)를 개발하여 기존에 개발된 QQ 미생물 구형담체 (QQ-beads)와 생물막오염 제어 성능을 QQ 활성과 물리세정 효과 측면에서 비교하였다. 동일 담체 부피 내에서 QQ 미생물 판형담체는 QQ 미생물 구형담체보다 QQ 활성에서 약 2.5배 뛰어났고 이러한 QQ활성은 담체의 표면적에 비례하는 것이 확인되었다. 또한, 중공사모듈의 바깥쪽에 위치하는 분리막에만 물리세정효과를 보인 구형담체와 달리, 판형담체는 안쪽과 바깥쪽에 위치하는 분리막 모두 고르게 물리세정 효과를 보였는데, 이는 판형담체가 중공사모듈 안쪽까지 자유롭게 침투가 가능하기 때문이다. 다발형의 중공사모듈이 설치된 연속식 MBR의 운전에서 QQ 미생물 판형담체는 기존에 개발된 QQ 미생물 구형담체에 비해 보다 뛰어난 QQ 활성과 물리세정효과로 인해 약 1.8배가량 생물막오염을 더 지연할 수 있음을 확인하였다. 둘째, MBR에서 생물막오염의 한 요소인 셀룰로오스를 분해하는 셀룰라아제 효소를 MBR에 적용하여 생물막오염 억제 가능성을 확인하였다. 활성슬러지의 생물막 내 셀룰로오스의 존재를 확인하였고 셀룰라아제는 활성슬러지의 생물막 형성을 효과적으로 제어함을 확인하였다. 셀룰라아제 생산 미생물인 Undibacterium sp. DM-1을 MBR 내 활성슬러지에서 분리, 동정하였다. 셀룰로오스 분해 미생물인 DM-1 미생물을 고정한 구형담체를 MBR에 적용하였고, 미생물이 고정되어 있지 않은 구형담체가 적용된 MBR에 비해 생물막오염 억제 효과가 약 2.2배 나타남을 확인하였다.Although a membrane bioreactor (MBR) has been widely applied for advanced wastewater treatment over the past two decades, membrane biofouling (i.e., biofilm formation on the membrane surface) still remains a major drawback that limits the widespread use. Recently, quorum quenching (QQ) has emerged as an effective biological control strategy for membrane biofouling in MBR. In particular, the use of QQ bacteria entrapping media (QQ-media) was proven to be efficient and economically feasible biofouling control in MBR. However, few studies have been conducted to explore how to increase the performance of QQ-media for biofouling control in MBR with different membrane types. In addition, because QQ is not effective in biofouling control after biofilm was formed, further studies are required to develop a new bacterium targeting degradation of already formed biofilm. In this study, QQ bacteria entrapping sheets (QQ-sheets) were developed as a new shape of moving QQ-media for alleviating in biofouling in MBR with a hollow fiber module. Moreover, cellulolytic bacteria were applied to mitigate biofouling in MBR by degrading cellulose-induced biofilm as an alternative biological control strategy to QQ-based control. Firstly, QQ-sheets as a new shape of moving QQ-media were developed to overcome the limitation of previously reported QQ-beads, particularly in MBR with a hollow fiber (HF) module. In a lab-scale MBR, QQ-sheets with a thickness of 0.5 mm exhibited a greater physical washing effect than did QQ-beads with a diameter of 3.5 mm because the former collided with membrane surfaces at the inner as well as the outer part of HF bundles, whereas the latter only made contact with the outer part. Moreover, QQ-sheets showed 2.5-fold greater biological QQ activity than did QQ-beads due to their greater total surface area at a fixed volume of QQ-media. Taking into account dense structure of HF bundles, these combined merits of QQ-sheets bring the QQ technology to practical applications in MBRs with commercial HF modules. Secondly, cellulase was introduced to MBR as a cellulose-induced biofilm control strategy. For practical application of cellulase to MBR, a cellulolytic (i.e., cellulose-degrading) bacterium, Undibacterium sp. DM-1, was isolated from a lab-scale MBR for wastewater treatment. Prior to its application to MBR, it was confirmed that the cell-free supernatant of DM-1 was capable of inhibiting biofilm formation and of detaching the mature biofilm of activated sludge and cellulose-producing bacteria. This suggested that cellulase could be an effective anti-biofouling agent for MBRs used in wastewater treatment. Undibacterium sp. DM-1 entrapping beads (i.e., cellulolytic-beads) were applied to a continuous MBR to mitigate membrane biofouling 2.2-fold, compared to an MBR with vacant-beads as a control. Subsequent analysis of cellulose content in biofilm formed on the membrane surface revealed that this mitigation was associated with an approximately 30% reduction in cellulose by cellulolytic-beads in MBR.Chapter I 1 I.1. Backgrounds 3 I.2. Objectives 5 Chapter II 7 II.1. Membrane Bioreactor (MBR) 9 II.1.1. Overview: MBR for Advanced Wastewater Treatment 9 II.1.2. Membrane Modules 18 II.1.3. Trend in MBR Market 24 II.1.4. Membrane Fouling in MBR 27 II.1.5. Fouling Control in MBR 32 II.2. Quorum Sensing (QS) 39 II.2.1. Definition and Mechanism 39 II.2.2. Mechanism 41 II.2.2.1. Gram-Negative Bacteria QS 41 II.2.2.2. Gram-Positive Bacteria QS 47 II.2.2.3. Interspecies QS communication 49 II.2.2.4. Other QS System 51 II.2.3. Role of QS in Biofilm Formation 56 II.2.4. Detection of AHL Signal Molecules 60 II.3. Quorum Quenching (QQ) 66 II.3.1. QS Control Strategy 66 II.3.1.1. Blockage of AHL Synthesis 68 II.3.1.2. Interference with Signal Receptors 70 II.3.1.3. Degradation of AHL Signal Molecules 71 II.3.2. Application of QQ to Control Biofouling in Membrane Process 76 II.3.2.1. Enzymatic QQ Application 76 II.3.2.2. Bacterial QQ 81 II.4. Immobilization Technique for biocatalyst 95 II.4.1. Whole-Cell Immobilization Method 95 II.4.2. Hydrogel 98 II.4.3. Nanofiber (Electrospun) 101 II.5. Extracellular Polymeric Substances (EPS) 106 II.5.1. Role of EPS in Biofilm Matrix: House of Biofilm Cells 106 II.5.2. Polysaccharides: Key Elements of EPS for Biofilm Formation 108 II.5.3. Control of Membrane Biofouling by Disruption of EPS 113 Chapter III 119 III.1. Introduction 121 III.2. Materials and Methods 123 III.2.1. Preparation of QQ-media 123 III.2.2. Fabrication of Hollow Fiber Modules 126 III.2.3. Fabrication of Polyacrylic Stick Modules 126 III.2.4. Assessment of Physical Washing Effect 128 III.2.5. Assessment of QQ Activity 129 III.2.6. MBR Operation 130 III.2.7. Analytical Methods 133 III.3. Results and Discussion 134 III.3.1. Comparison of QQ Efficiency using QQ-beads between Single- and Multi-layer HF Modules 134 III.3.2. Development of Sheet-shaped Media 139 III.3.3. Evaluation of Biofouling Control by QQ-sheets in MBRs with Single- and Multi-layer HF Modules 148 III.3.4. Direct Comparison of Biofouling Mitigation between QQ-sheets and QQ-beads in MBR with HF Module 152 III.4. Conclusions 154 Chapter IV 155 IV.1. Introduction 157 IV.2. Material and Methods 159 IV.2.1. Bacterial Strains and Culture Conditions 159 IV.2.2. Visualization of Cellulose and Biofilms of Activated Sludge 159 IV.2.3. Isolation of Cellulolytic Microorganisms and Cellulose-producing Bacteria from MBR 161 IV.2.4. Assay for Biofilm Formation and Detachment 164 IV.2.5. Preparation of Beads for MBR Application 165 IV.2.6. Biostability of Cellulolytic-beads in MBR 166 IV.2.7. MBR Operation 166 IV.2.8. Analysis of Cellulose and EPS in Biofilm in MBR 169 IV.3. Results and Discussion 170 IV.3.1. Effect of Cellulase on Biofilm Formation of Activated Sludge in MBR 170 IV.3.2. Isolation and Identification of Cellulolytic Microorganism 174 IV.3.3. Anti-biofouling Activity of Undibacterium sp. DM-1 176 IV.3.4. Effect of Cellulolytic-beads on Mitigation of Membrane Biofouling 180 IV.3.5. The Correlation between Cellulose, EPS and Membrane Biofouling in MBR 182 IV.3.6. Biostability of Cellulolytic-beads 187 IV.4. Conclusions 190 Chapter V 191 국문초록 195 Reference 197Docto