[A-HW24] Material transportation and cycling at the land-sea interface: from headwaters to the ocean

[AHW24-P01] Shotgun metagenomic analysis on the groundwater microbial communities and screening of the functional genes related to the nitrogen cycling in Ryukyu Limestone area

*Maruyama Rio¹, Nanami Mizusawa¹, Jun Yasumoto², Mariko Iijima³, Yasumoto Ko¹, Mina Hirose⁴, Akira Iguchi³, Mitsuru Jimbo¹, Shugo Watabe¹, Takada Ryogo⁵, Takahiro Hosono⁶, Oktanius Richard Hermawan⁶ (1.Kitasto Univercity, 2.Faculty of Agriculture University of the Ryukyus, 3.National Institute of Advanced Industrial Science and Technology (AIST), 4.Tropical Technology Plus, 5.Center for Strategic Research Projects, University of the Ryukyus, 6.Faculty of Advanced Science and Technology, Kumamoto University)

Keywords:shotgun metagenomics, microbial communities, groundwater

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 groundwaters of the Okinawajima 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. Environmental factors were also measured at the same time. Approximately 1 L of groundwater samples were collected from three locations, G5, G23 and M8E07, 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 were 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. The shotgun analysis showed that bacteria accounted for 90-98 % in the microbial communities including archea, eukaryotes and viruses for all the sampling locations. At the G23 site, Oscillatoriophycideae and Nostocales both belonging to Cyanobacteria accounted for 41 % and 15 % in the microbial community, respectively. These cyanobacteria were rarely found in the other two sites, suggesting that the G23 site provided an environment suitable for the growth of these cyanobacteria. Nitrosopumilales and Nitrospira, which belong to archea and bacteria respectively and are known to contain strains involved in the nitrogen cycling, appeared at appreciable ratios only in the M8E07 site, suggesting that this site had a reductive environment. Interestingly, the M8E07 contained much higher denitrification genes, nirK and nosZ, and much lower nitrogen fixation genes, nifD, nifK and nifH, than the other two sites. Further studies are needed to elucidate how the nitrogen-cycling genes in the microbial communities are related to aquatic environmental factors.