[A-HW18] Material transportation and cycling at the land-sea interface: from headwaters to the ocean

[AHW18-P20] Microbial Communities and Genes Related to Nitrogen and Sulfur Metabolisms in the Ryukyu Limestone Aquifer

*Maruyama Rio¹, Nanami Mizusawa¹, Ko Yasumoto¹, Ryogo Takada², Jun Yasumoto³, Mina Yasumoto⁴, Mariko Iijima⁵, Iguchi Akira⁵, Okutanius Richard Hermawan⁶, Takahiro Hosono^7,8, KE-HAN SONG⁹, Ryuichi Shinjo^9,10, Shugo Watabe¹ (1.Kitasto Univercity, 2.Center for Strategic Resarch Projects, University of Ryukyus, 3.Faculty of Agriculture, University of the Ryukyus, 4.Tropical Technology Plus, 5.National Institute of Advanced Industrial Science and Technology (AIST), 6.Earth and Environmental Science, Graduate School of Science and Technology,, 7.Faculty of Advanced Science and Technology, Kumamoto University,, 8.International Research Organization for Advanced Science and Techmology, Kumamoto University, 9.Department of Physics and Earth Sciences, University of the Ryukyus, 10.Resarch Institute for Humanity and Nature)

Keywords:Groundwater, Microbial communities, Nitrogen metabolism, Sulfer metabolism

Microbial communities are thought to play important roles in the material cycling in ecosystems. However, corresponding data in groundwater are hardly available at present, although the groundwater is a valuable water resource in island regions where rivers are not abundant enough. Nevertheless, groundwater contamination by nitrate-nitrogen (NO₃-N) has become a problem in recent years in these regions. In this study, we analyzed the microbial communities in the Ryukyu limestone aquifer using shotgun metagenomics, and determined functional genes involved in nitrogen and sulfur metabolisms. We also measured environmental factors and verified the relationship of the above-mentioned microbial communities with functional genes.
Groundwater samples were collected monthly from November 2021 to July 2022 at an observation well in the southern region of Okinawa Prefecture, and environmental factors were measured. 1 L of collected groundwater were filtered through a Sterivex 0.22 µm filter (Millipore) and the filters were frozen at -20 °C until DNA extraction. After thawing the filter samples, DNA extraction was performed using DNeasy Power Water Sterivex kit (QIAGEN), and shotgun libraries were prepared using Nextra XT DNA Preparation Kit (Illumina). Sequences obtained with a MiSeq next-generation sequencer (Illumina) were processed using FLASH and FASTX-Toolkit, and the microbial communities and functional genes were analyzed using BLASTX searches.
NO₃-N levels exceeded the drinking water standard of 10 mg/L in November 2021 and June 2022. Sulfate ion (SO₄^2-) concentrations were also high in these months. Meanwhile, the content of suspended solids (SS) was extremely high in March and April 2022. The yield of filter-trapped DNA was low in November 2021 and March-June 2022, and high from December 2021 to February 2022 and July 2022.
Different trends in the microbial communities were observed during these two periods consisting of December 2021-February 2022 and March 2022-June 2022, respectively. Alphaproteobacteria were abundant in the overall field survey period. It was noted that Gammaproteobacteria and Flavobacteria were more abundant in December 2021-February 2022 than in March 2022-June 2022. On the other hand, Actinomycetia, Planctomycetes and Acidobacteria were more abundant in the latter than in the former. Shannon's diversity index was high from December 2021 to February 2022, and low in November 2021 and March to May 2022. Acidobacteria is known to be a soil-derived bacterium. Furthermore, the increase in SS levels in March and April 2022 suggests that the soil was introduced into the groundwater in these months.
The screening using the metagenomic data demonstrated that genes related to nitrate reduction, denitrification, and anabolic sulfur metabolisms were markedly more abundant from December 2021 to February and in July 2022 than in the other months, the trend of which was similar to that of the yield of recovered DNA. These results suggest that the aforementioned metabolisms were enhanced from December 2021 to February 2022, resulting in the decrease of NO₃-N and SO₄^2- amounts. Interestingly, when the amount of recovered DNA, which is thought to be proportional to the abundance of microbes, was high, the diversity of the microbial communities was also high. In conclusion, it is suggested in the Ryukyu limestone aquifer that the increase in the SS content triggered the decrease in both microbial abundance and diversity, resulting in the increase in NO₃-N and SO₄^2- amounts.