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.