Microbial communities and functional genes involved in the nitrogen cycling of groundwater in the Ryukyu Limestone area as revealed by shotgun metagenomics

While microbial biogeochemical activities such as those involving denitrification and sulfate reduction have been considered to play important roles in the material cycling in various aquatic ecosystems, our current understanding of the microbial community in the groundwater ecosystem is strikingly insufficient. As a first step to assess the groundwater of the Okinawa-jima locating in the southernmost region of Japan, we performed shotgun metagenomic analysis on their microbial communities, and screened the functional genes involved in the nitrogen cycling. Approximately 1 L of the groundwater samples were collected from three locations, Sites 1, 2 and 3, in Yaese Town characterized by a high agricultural activity. Water samples were filtered through a Sterivex filter (QIAGEN) with a pore size of 0.22 µm and cells trapped on the filter were stored frozen at -20 ℃ until DNA extraction. DNA extraction from the Sterivex filters was performed using DNeasy Power Water Sterivex kit (QIAGEN). Then, shotgun libraries were prepared using the Nextra XT DNA Preparation Kit (Illumina). Metagenomic analysis was performed using a MiSeq next-generation sequencer (Illumina). The obtained nucleotide sequences were preprocessed using FLASH and FASTX-Toolkit, and then analyzed for the microbial community and functional gene composition by BLASTX search. Environmental factors were also measured at the same time when the samples were collected.The shotgun analysis showed that bacteria accounted for 90-98 % in the microbial communities including archea, eukaryotes and viruses for all the sampling locations. The bacterial communities involving the nitrogen cycling shifted by month in each site, indicating that this cycle was accomplished not with specific bacteria, but rather with the integration of the bacterial community. Interestingly, Site 3 contained much higher denitrification genes, narG/narH/nxrB, narG/narZ/nxrA, napA, nirK, norB and nosZ, than the other two sites. This site was thought to have undergone the denitrification reaction with relative high amounts of dissolved organic carbon (DOC). While Site 2 was characterized by a high nitrate-nitrogen content and a low amount of DOC, this site provided a moderate amount of denitrification genes. Site 1 showed markedly lower amounts of all nitrogen-cycle genes.