동해 울릉분지 퇴적물 내 혐기성 유기탄소 분해에 관련된 미생물 군집구조 = Microbial community structures associated with anaerobic carbon oxidation in the Ulleung Basin sediments, East Sea

다국어 초록 (Multilingual Abstract)

A combination of metabolic measurements and molecular microbiological analyses using 16S rRNA-stable isotope probing (RNA-SIP) method was used to investigate the microbial communities responsible for the oxidation of acetate, the terminal electron donor in anaerobic carbon oxidation processes, in the costal continental shelf (EB1 site) and center of the basin (EB6) sediment of the Ulleung Basin (UB). Time-course incubation experiments with the addition of acetate revealed that microbial iron, and nitrate reduction were simultaneously responsible for acetate oxidation at the EB1 site. Whereas manganese oxide reduction was responsible for main acetate oxidation in the slurries amended with acetate of the basin during incubations. Active bacterial 16S rRNA of members of the genera Arcobacter within the Epsilonproteobacteria appeared to be a predominant acetate oxidizer associated with nitrate and iron reduction, but they were not responsible for manganese reduction in the slurries from the continental shelf. Repeated addition of organic carbons would trigger anaerobic nitrification to nitrate to support denitrification by acetate-oxidizing Arcobacter during anaerobic slurry incubation. Manganese reducers in affiliated with the Colwellia and Oceanospirillaceae in the gammaproteobacteria were main acetate oxidizer in acetate-amended slurries of EB6. In acetate-amended slurries from each site, the majority of archaeal members labelled with 13C-acetate belonged to Marine Group I Archaea (MGI), which adapt to anaerobic condition and may utilize added acetate for a living, thus they may play a significant role in carbon and nitrogen cycles. Overall, our results provide new metabolic information on denitrification coupled to anaerobic nitrification in organic carbon enriched conditions.

Heterotrophic anaerobic nitrification was observed in slurries repeatedly amended with organic carbon source during anaerobic incubations of the sediment of UB. Subsequently, the production of nitrate supported the nitrate reduction by Arcobacter over several days under anaerobic conditions. Heterotrophic anaerobic nitrification may be coupled to the reduction of manganese or iron oxide, which would be stimulated by the addition of organic carbon. Similarly, heterotrophic anaerobic nitrification triggered by acetate was observed during anaerobic incubation from slurry experiments of intertidal mudflat sediment of the Ganghwa Island where iron oxide appeared as high concentration (40 μmol cm-3). Meanwhile, MGI archaea, a significant ammonia oxidizer in marine environments, appeared to be the 13C-labelled archaeal group in acetate-amended slurry (in Chapter 2). As overall results, I speculate that MGI archaea would be concerned in anaerobic nitrification. In Chapter 3, I propose a study to determine the ability of anaerobic nitrification of MGI in anaerobic organic matter rich-sediment and to explore their role in the ecosystem.

We investigated the biogeochemical constituents, microbial communities and functional genes (mcr and dsr) associated with anaerobic methane oxidation and sulfate reduction, and metabolic activities by sulfate reduction in the sulfate-methane transition zone (SMTZ) of gas hydrate-bearing sediment of the Ulleung Basin in the East Sea. Maxima in the sulfate reduction rate (12.6 nmol cm-3 d-1), CO concentration (83 μM), and gene abundances of dsrA (9.1×106 copies cm-3) and mcrA (11.6×106 copies cm-3) occurred in the SMTZ. The peaks of CO consistently found in the SMTZ suggested that CO is an intermediate metabolic product related to methane oxidation. Candidate division JS1, the predominant bacterial group that comprised 59.0–63.7% of 16S rRNA gene sequences, was recognized as an important organic carbon oxidizer. Both Marine Benthic Group D (MBGD) and Marine Benthic Group B (MBGB), which constituted 40.8–52.9 and 10.3–43.9% of 16S rRNA gene sequences, respectively, were the dominant archaeal groups. Analysis of functional gene diversity revealed that ANME-1-related phylotypes appeared to be the major CH4 oxidizer, whereas Firmicute-like group was a predominant sulfate reducer in the 0.8 mbsf in SMTZ with low SO42- concentration. Overall results indicated that JS1 and two archaeal groups (MBGB and MBGD) seem to play a significant role in carbon and elements cycles in the gas hydrate-bearing subsurface sediment of the Ulleung Basin.

목차 (Table of Contents)

• Contents
• LIST OF TABLES V
• LIST OF FIGURES VI
• ABSTRACT X
• Chapter 1. General introduction 1
• 1. Introduction 2
• 1.1. Role of bacteria in biogeochemical cycles of marine sediments 3
• 1.1.1. Denitrification 4
• 1.1.2. Manganese reduction 5
• 1.1.3. Iron reduction 6
• 1.1.4. Sulfate reduction 7
• 1.1.5. Methanogenesis 8
• 2. East Sea 9
• 2-1. Geochemical properties in sediment of Ulleung Basin 9
• 2.2. Composition of Prokaryotes in the sediment of East Sea 14
• 2.2.1. Culture-dependent prokaryotes 14
• 2.2.2. Culture-independent prokaryotes 15
• 2.2.3. Study of microbial community in the sediments of UB 16
• 3. Outline of this thesis 18
•  
• Chapter 2. Acetate oxidizing microbial communities revealed by RNA-stable isotope probing in the continental shelf and center of basin sediment of the Ulleung Basin, East Sea 20
• 1. Introduction 21
• 2. Materials and methods 25
• 2.1. Study site, sediment sampling, and handling 25
• 2.2. Anaerobic total carbon oxidation and iron- and sulfate- reduction rates 25
• 2.3. Slurry incubations 27
• 2.4. Porewater and solid-phase Fe analyses 28
• 2.5. RNA/DNA extraction and density gradient centrifugation 30
• 2.6. Terminal restriction fragment length polymorphism, cloning, sequencing and phylogenetic analysis 30
• 3. Results 32
• 3.1. Geochemical properties of the continental shelf and basin of the UB 32
• 3.2. Partitioning of C oxidation of the continental shelf and the center of basin 33
• 3.3. Slurry incubation of the continental shelf sediment 38
• 3.4. Slurry incubation of the basin center sediment 40
• 3.5. 13C-labelled microbial groups revealed by stable isotope probing 42
• 3.6. Microbial diversity of the original sediments of the continental shelf and the center of basin 47
• 3.7. Diversity of 13C-labelled bacterial 16S rRNA genes 54
• 3.8. Diversity of 13C-labelled archaeal 16S rRNA genes 56
• 4. Discussion 57
• 4.1. Carbon oxidation pathways in the sediment of UB 57
• 4.2. Acetate oxidizing bacteria 58
• 4.3. Putative ammonia oxidizing archaea 62
• 4.4. Putative anaerobic nitrification coupled to denitrification by Arcobacter 64
• Chapter 3. Heterotrophic anaerobic nitrification stimulated by addition of organic carbon in marine sediment. 67
• 1. Background 68
• 2. Materials and methods 70
• 2.1. Slurry incubation 70
• 2.2. DNA extraction, quantification PCR of amoA gene 71
• 3. Results and discussion 73
• 3.1. Geochemical evidence of heterotrophic anaerobic nitrification 73
• 3.2 Heterotrophic anaerobic nitrification in the sediments of the continental shelf and the center of basin 74
• 3.3. Stimulation by addition of organic carbon 75
• 3.4. Who are responsible for heterotrophic anaerobic nitrification? 82
• Chapter 4. Microbial community structure associated with biogeochemical processes in the sulfate-methane transition zone (SMTZ) of gas hydrate-bearing sediment of the Ulleung Basin, East Sea 85
• 1. Introduction 86
• 2. Materials and Methods 89
• 2.1. Site description and sampling 89
• 2.2. Geochemical analyses 91
• 2.3 Sulfate reduction rates 91
• 2.4. Total microbial biomass 92
• 2.5. DNA extraction and quantification of dsrA and mcrA genes 92
• 2.6. Diversity of 16S rRNA, and the dsrAB, and mcrA genes 93
• 2.7. Nucleotide sequence accession numbers 96
• 3. Results 97
• 3.1 Geochemical parameters 97
• 3.2. Microbial biomass and sulfate reduction activity 99
• 3.3. Abundance of dsrA and mcrA genes 101
• 3.4. Microbial community composition in gas hydrate-bearing sediments 101
• 3.5. Archaeal community composition in gas hydrate bearing sediments 108
• 3.6. Characterization of dsrAB and mcrA sequence distributions within the SMTZ 110
• 4. Discussion 114
• 4.1. Bacterial communities in gas hydrate bearing sediments of the UB 114
• 4.2. Archaeal community structure related to anaerobic oxidation of methane 116
• 4.3. Microbial biogeochemistry in the SMTZ of the UB sediment 117
• Chapter 5. General conclusions 119
• 1. General conclusions 120
• REFERENCES 124
• 국문요지 146
• Appendix 149