Shotgun metagenomic analysis of the microbial communities and associated network of nitrogen metabolism genes in the Ryukyu limestone aquifer

Abstract

While microbial biogeochemical activities such as those involving denitrification and sulfate reduction have been considered to play important roles in material cycling in various aquatic ecosystems, our current understanding of the microbial community in groundwater ecosystems is remarkably insufficient. To assess the groundwater from three locations in the Ryukyu limestone aquifer of Okinawa Island, which is located in the southernmost region of Japan, we performed shotgun metagenomic analysis on the microbial communities at the three sites and screened for functional genes associated with nitrogen metabolism. Shotgun analysis showed that bacteria accounted for 90–98% of the microbial communities, which included archaea, eukaryotes and viruses, at all three sampling sites. The bacterial communities associated with nitrogen metabolism shifted by month at each site, indicating that this metabolism was accomplished not via specific bacteria but rather by the bacterial community as a whole. Interestingly, site 3 contained much higher levels of the 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 denitrification that was driven by high quantities of dissolved organic carbon (DOC). In contrast, site 2 was characterized by a high nitrate-nitrogen (NO3-N) content and a low amount of DOC, and this site yielded a moderate amount of denitrification genes. Site 1 showed markedly low amounts of all nitrogen metabolism genes. Overall, nitrogen metabolism in the Ryukyu limestone aquifer was found to change based on environmental factors.