10:45 AM - 12:15 PM
[AHW18-P22] Characteristics of nirS denitrification-related genes variation in groundwater in the Ryukyu limestone area
Keywords:Groundwater, nirS denitrification-related genes, Real-Time PCR
Although groundwater contamination by nitrate-nitrogen (NO3-N) has emerged as a global concern, the quantity of inorganic nitrogen eliminated by denitrification in the nitrogen cycle remains uncertain. Accurate estimation of the amount of nitrogen removal by denitrification in the nitrogen cycle is vital for sustainable use and management of groundwater. Understanding the quantity of microorganisms engaged in denitrification reactions is an effective approach to achieving this goal. Recently, genetic analysis using nitrite reductase (nirK and nirS) genes as markers has been developed to detect microorganisms involved in denitrification reactions. However, studies on its application to groundwater are scarce.
In this study, we aimed to quantify the amount of microorganisms involved in denitrification reactions in groundwater by using the nitrite reductase gene nirS and to understand their fluctuating characteristics. Groundwater quality data and microbiota analysis were conducted using qPCR in the Ryukyu limestone aquifer in the southern Okinawa Island region. Among the observation wells distributed in the southern area of Okinawa Island, we selected three sites (St.1 to St.3), including the site where the denitrification reaction was confirmed, and conducted groundwater sampling periodically once a month from November 2021 to July 2022. Samples for genetic analysis were collected in sterile bottles, refrigerated, and brought back to the laboratory. One liter of water sample was subjected to suction filtration using a Steribex filter in the laboratory, and DNA was extracted from the filtration filter. The 16S rRNA gene and nitrite reductase gene nirS were quantified by qPCR. Samples for water quality analysis were filtered with a 0.2 μm filter on-site, refrigerated, and brought back to the laboratory. Major dissolved ions were measured using ion chromatography; dissolved organic carbon (DOC) was analyzed using a TOC meter, and total phosphorus (TP) and dissolved phosphorus (DP) were analyzed using a colorimetric method (MB method).
Results showed that the nirS gene in groundwater had a median value of 3.47 x 104 copies/mL, a maximum value of 2.01 x 105 copies/mL, and a minimum value of 7.20 x 103 copies/mL at site St. 3, where the denitrification reaction was confirmed. The nirS gene was high during periods when NO3-N concentrations in groundwater were high and tended to be low during periods when NO3-N concentrations were decreasing due to denitrification reactions. Furthermore, nirS gene showed a strong positive correlation with NO3-N and K+ and a moderate positive correlation with water temperature, Cl-, SO43-, Mg2+, and DOC. On the other hand, dissolved oxygen (DO) showed a negative and weak correlation with the nirS gene.
These results indicate that the nirS gene amount is augmented by the increase of fertilizer components such as NO3-N and electron donors, the increase of water temperature, and the formation of an anaerobic environment due to denitrification reactions. To conclude, this study led to the understanding of the formation of an anaerobic environment due to denitrification reactions, the amount of microbes involved in denitrification reactions in groundwater and related variable characteristics.
In this study, we aimed to quantify the amount of microorganisms involved in denitrification reactions in groundwater by using the nitrite reductase gene nirS and to understand their fluctuating characteristics. Groundwater quality data and microbiota analysis were conducted using qPCR in the Ryukyu limestone aquifer in the southern Okinawa Island region. Among the observation wells distributed in the southern area of Okinawa Island, we selected three sites (St.1 to St.3), including the site where the denitrification reaction was confirmed, and conducted groundwater sampling periodically once a month from November 2021 to July 2022. Samples for genetic analysis were collected in sterile bottles, refrigerated, and brought back to the laboratory. One liter of water sample was subjected to suction filtration using a Steribex filter in the laboratory, and DNA was extracted from the filtration filter. The 16S rRNA gene and nitrite reductase gene nirS were quantified by qPCR. Samples for water quality analysis were filtered with a 0.2 μm filter on-site, refrigerated, and brought back to the laboratory. Major dissolved ions were measured using ion chromatography; dissolved organic carbon (DOC) was analyzed using a TOC meter, and total phosphorus (TP) and dissolved phosphorus (DP) were analyzed using a colorimetric method (MB method).
Results showed that the nirS gene in groundwater had a median value of 3.47 x 104 copies/mL, a maximum value of 2.01 x 105 copies/mL, and a minimum value of 7.20 x 103 copies/mL at site St. 3, where the denitrification reaction was confirmed. The nirS gene was high during periods when NO3-N concentrations in groundwater were high and tended to be low during periods when NO3-N concentrations were decreasing due to denitrification reactions. Furthermore, nirS gene showed a strong positive correlation with NO3-N and K+ and a moderate positive correlation with water temperature, Cl-, SO43-, Mg2+, and DOC. On the other hand, dissolved oxygen (DO) showed a negative and weak correlation with the nirS gene.
These results indicate that the nirS gene amount is augmented by the increase of fertilizer components such as NO3-N and electron donors, the increase of water temperature, and the formation of an anaerobic environment due to denitrification reactions. To conclude, this study led to the understanding of the formation of an anaerobic environment due to denitrification reactions, the amount of microbes involved in denitrification reactions in groundwater and related variable characteristics.