Metagenomic analysis of the microbial communities and associated network of nitrogen metabolism genes in the Ryukyu limestone aquifer.

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 in the Ryukyu limestone aquifer of Okinawa Island, which is located in the southernmost region of Japan, we performed metagenomic analysis on the microbial communities at the three sites and screened for functional genes associated with nitrogen metabolism. 16S rRNA amplicon analysis showed that bacteria accounted for 94-98% of the microbial communities, which included archaea at all three sites. The bacterial communities associated with nitrogen metabolism shifted by month at each site, indicating that this metabolism was accomplished by the bacterial community as a whole. Interestingly, site 3 contained much higher levels of the denitrification genes such as narG and napA 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.

Mechanism of denitrification in subsurface-dammed Ryukyu limestone aquifer, southern Okinawa Island, Japan.

Denitrification crucially regulates the attenuation of groundwater nitrate and is unlikely to occur in a fast-flowing aquifer such as the Ryukyu limestone aquifer in southern Okinawa Island, Japan. However, evidences of denitrification have been observed in several wells within this region. This study analyzed environmental isotopes (δ15NNO3 and ẟ18ONO3) to derive the rationale for denitrification at this site. Additionally, the presence of two subsurface dams in the study area may influence the processes involved in nitrate attenuation. Herein, we analyzed 150 groundwater samples collected spatially and seasonally to characterize the variations in the groundwater chemistry and stable isotopes during denitrification. The values of δ15NNO3 and δ18ONO3 displayed a progressive trend up to +59.7 ‰ and + 21 ‰, respectively, whereas the concentrations of NO3--N decreased to 0.1 mg L-1. In several wells, the enrichment factors of δ15NNO3 ranged from -6.6 to -2.1, indicating rapid denitrification, and the δ15NNO3 to δ18ONO3 ratios varied from 1.3:1 to 2:1, confirming the occurrence of denitrification. Denitrification intensively proceeds under conditions of depleted dissolved oxygen concentrations (<2 mg L-1), sluggish groundwater flow with longer residence times, high concentrations of dissolved organic carbon (>1.2 mg L-1), and low groundwater levels during the dry season with precipitation rates of <100 mm per month (Jun-Sep). SF6 analysis indicated the exclusive occurrence of denitrification in specific wells with groundwater residence times exceeding 30 years. These wells are located in close proximity to the major NE-SW fault system in the Komesu area, where the hydraulic gradient was below 0.005. Detailed geological and lithological investigations based on borehole data revealed that subsurface dams did not cause denitrification while the major NE-SW fault system uplifted the impermeable basement rock of the Shimajiri Group, creating a lithological gap at an equivalent depth that ultimately formed a sluggish groundwater area, promoting denitrification.