Title Page
ABSTRACT
국문 초록
PREFACE
Contents
CHAPTER 1. Introduction 21
1.1. Nitrate contamination of water 22
1.2. Biological nitrate removal 25
1.3. Biofilm-based water treatment processes 28
1.4. Hydrogen-based membrane biofilm reactor 30
1.5. The objective of this study 35
CHAPTER 2. Effects of hydrogen pressure for stabilization with improved denitrification in a hydrogen-based membrane biofilm reactor 37
2.1. Abstract 38
2.2. Introduction 41
2.3. Materials and methods 45
2.3.1. Experimental setup 45
2.3.2. Determination of H₂ permeability 48
2.3.3. Flux calculation and chemical analysis 52
2.3.4. Preparation of the inoculum and influent water 53
2.3.5. Experimental conditions 54
2.3.6. Analytical methods 55
2.4. Results and discussion 56
2.4.1. H₂ permeability of the membrane 56
2.4.2. Effect of H₂ pressure on change in denitrification rate of the inoculum 59
2.4.3. Effect of H₂ pressure on biomass attachment on membrane surfaces 64
2.4.4. Effect of H₂ pressure on biomass attachment on membrane surfaces 70
2.4.5. Effect of H₂ pressure on biomass attachment on membrane surfaces 74
2.4.6. Practical implications 77
2.5. Conclusions 80
2.6. Supplementary materials 82
CHAPTER 3. Biofilm characteristics for providing resilient denitrification in a hydrogenbased membrane biofilm reactor 85
3.1. Abstract 86
3.2. Introduction 88
3.3. Materials and methods 92
3.3.1. Preparation of synthetic groundwater and inoculation 92
3.3.2. Laboratory-scale experimental setup and chemical analyses 93
3.3.3. Calculations 94
3.3.4. Operating conditions 96
3.3.5. Biofilm sampling and microbial analysis 99
3.4. Results and discussion 101
3.4.1. Removal patterns of multiple electron acceptor according to phases 101
3.4.2. Resilience of denitrification 107
3.4.3. Characteristics of microbial community in biofilms 111
3.4.4. Implication of a physically thick biofilm 116
3.5. Conclusions 121
3.6. Supplementary materials 122
CHAPTER 4. Genome-resolved metagenomic reveals contribution of heterotrophic denitrification in a hydrogen-based membrane biofilm reactor 129
4.1. Abstract 130
4.2. Introduction 132
4.3. Materials and methods 135
4.3.1. H₂-MBfR operation 135
4.3.2. Shotgun sequencing and library construction 136
4.3.3. Functional gene annotation, quantification of gene abundance, and MAG construction 137
4.4. Results and discussion 139
4.4.1. Features of oxyanions removal 139
4.4.2. Metagenomic pathways and related genes of interest 143
4.4.3. Distribution of genes of interest in MAGs 150
4.4.4. Comparison of gene abundance by taxonomic group 154
4.4.5. MAGs associated with homoacetogens 157
4.5. Conclusions 162
3.6. Supplementary materials 164
CHAPTER 5. Heterotrophs providing resilience to hydrogen supply disturbances in a hydrogen-based membrane biofilm reactor 167
5.1. Abstract 168
5.2. Introduction 169
5.3. Materials and methods 173
5.3.1. Experimental setup 173
5.3.2. Inoculation procedure 176
5.3.3. Operating conditions 176
5.3.4. Analytical methods and flux calculation 177
5.4. Results and discussion 179
5.4.1. Denitrification performance 179
5.5. Conclusions 185
CHAPTER 6. Conclusions 186
6.1. Summary of the thesis 187
6.2. Significance 189
References 190
Table 1.1. Summary of characteristics of nitrate removal technologies. 27
Table 2.1. The chemical composition of synthetic medium used for diluting activated sludge 53
Table 2.2. H₂ pressure and NO₃-N concentration by the operating phase 55
Table 2.3. Possible scenarios for biofilm accumulation including biomass acclimation and attachment, and implications based on the results of this study 79
Table 3.1. Summary of acceptor-substrate loadings and influent temperatures according to experimental phase 98
Table 3.2. Biofilm thicknesses reported in previous studies with the MBfR 119
Table 3.3. Comparison of maximum nitrate-nitrogen removal fluxes in published papers with the H₂-based MBfR 120
Table 4.1. Abundances of genes related to metabolic pathways such as H₂ oxidation, and NO₃¯, ClO₄¯ and SO₄²¯ reduction, and organic carbon metabolism 149
Table 4.2. MAGs associated with homoacetogens 160
Table 4.3. Electron equivalents for H₂ oxidation, acetate production, and oxidation 160
Figure 1.1. Hazardous effects of nitrate on human health and environment. Photographs of (a) a baby with cyanosis and (b) algal blooms spreads across... 24
Figure 1.2. Schematic of biofilm-based processes. (a) moving-bed biofilm reactor, (b) trickling filter reactor, and (c) fluidized-bed biofilm reactor. 29
Figure 1.3. Schematic diagram of denitrification in H₂-MBfR process. 33
Figure 1.4. Substrates gradient profiles from membrane lumen to bulk liquid in a counter-diffusion method. 34
Figure 2.1. Schematic of experimental setup for the continuous flow H₂-MBfR. 47
Figure 2.2. Schematic of the H₂-permeation test. (a) The experimental setup for H₂-permeation test and (b) A typical H₂ concertation profile from... 51
Figure 2.3. Change in headspace pressure during H₂-permeation test. The headspace pressure gradually increased and reached steady state at 0.32 bar... 58
Figure 2.4. Comparison of denitrification rate according to 1.1, 1.3, and 1.6 bar of H₂ pressure (absolute pressure). Changes in (a) the NO₃-N... 63
Figure 2.5. Comparison of biomass attachment to the membrane surfaces according to the H₂ pressure. Changes in (a) the mixed liquor suspended... 68
Figure 2.6. The result of microscopic observation. (a) The morphology of flocs and (b) the process by which flocs accumulate on the membrane... 69
Figure 2.7. Comparison of electron acceptor removal fluxes and TN removal of the H₂-MBfR varying biomass attachment by H₂ pressure during inoculation. After biomass attachment at 1.0, 1.3 and 1.6 bar of H₂, continuous... 73
Figure 2.8. Improvement of removal flux with increasing H₂ and NO₃-N loading. This is the result of the follow-up experiments on biomass... 76
Figure 3.1. Surface loading rates (top) and removal percentages of oxyanions (bottom) for all operating phases. 105
Figure 3.2. The e¯ eq removal fluxes and maximum H₂-delivery flux through the experimental phases. Removal fluxes are averages when NO₃-N removal... 106
Figure 3.3. Patterns of performance loss and recovery: effects of (a) increasing loading rates (phase 1-2 and end of 1-4), (b) pump malfunction... 110
Figure 3.4. Distribution of microbial taxa with relative abundance greater than 0.5% in the seed sludge and biofilm. The taxa are organized by known metabolic functions (Tables S3.2 and S3.3). DB, PRB, NB, SRB, and SOB... 115
Figure 4.1. (a) Comparison of loadings and removal fluxes of all electron acceptors. Electron acceptors included NO₃-N, ClO₄¯, SO₄₋S, and O₂.... 142
Figure 4.2. Metabolic pathways for the following reactions: (a) H2 oxidation and NO3-, ClO4- and SO42-reduction and (b) Organic carbon metabolism pathways including homoacetogenic reactions (red arrows). Each... 148
Figure 4.3. Heatmap describing gene abundance profile of the microorganisms with respect to H₂ oxidizing enzymes and reductases of NO₃¯, NO₂¯, SO₄²¯, and ClO₄¯. The TPM value of oxidase or reductases is the sum of the... 153
Figure 4.4. Sum of abundances of gene set of interest included in taxa affiliated with MAGs (a) at the class level, and (b) at the genus level belonging to Betaproteobacteria. 156
Figure 4.5. Schematic diagram of the overall microbial metabolism that would have occurred within the biofilm. 161
Figure 5.1. Schematic of laboratory scale experimental setup of the H₂-MBfR. 175
Figure 5.2. Comparison of denitrification performance trends of (a) R1 and (b) R2 for H₂ shocks. 181
Figure 5.3. Concentration of soluble chemical oxygen demand of effluent samples. 182
Figure 5.4. Comparison of normalized fluxes considering H2 shocks as individual phases. 183
Figure 5.5. Comparison of quantitative performance reduction along with H2 shocks. 184