Research on Microbial Community and Metabolic Function in Groundwater of High-level Radioactive Waste Disposal Underground Laboratory Site

Microorganisms have the potential to affect the long-term safety of geological disposal of high-level radioactive waste (HLW). They not only drive the biogeochemical processes in the deep environment but also have a direct impact on the safety performance of the disposal process. China is currently constructing its first underground research laboratory for HLW disposal—the Beishan Underground Research Laboratory (URL)—at the Xinchang site in Gansu Province. However, the microbial communities and their functions in the underground environment of the Beishan URL site remain poorly understood. In this study, groundwater samples were collected from representative water-conducting fractures at various depths along the Beishan URL ramp, and metagenomics was employed for the first time to reveal the microbial composition and functional profiles of groundwater in the Beishan URL site. The results are as follows, bacteria are dominant in the groundwater community of the Beishan URL site, and the diversity is significantly higher than that of fungi and archaea. Proteobacteria, Unclassified_d_Bacteria, Patescibacteria, Firmicutes and Actinobacteria are the dominant phyla, and Unclassified_d_Bacteria, Rugosibacter, HB2-32-21, MEYI01 and Methylophilus are the dominant genera. Correlation network analysis shows that K⁺ concentration and temperature are significantly correlated with multiple microbial phyla. Specifically, K⁺ concentration exhibits a positive correlation with multiple phyla, likely serving as an essential nutrient for microbial metabolism, whereas temperature shows a negative correlation, potentially inhibiting metabolic activities at elevated levels. These two factors may be key factors affecting the microbial community structure of groundwater in the Beishan URL site. Considering that the long-term safety of geological disposal of HLW depends on the stability of the site’s biogeochemical conditions, especially the microbial-driven methane production and sulfate reduction process, this study analyzed the functions of microbial methane metabolism pathway and sulfur metabolism pathway. Although genes associated with the acetoclastic methanogenesis pathway (e.g., ACSS1_2/acs, ackA) are widely present, the crucial methanogenic marker gene mcrA is completely absent. This absence may be attributed to redox conditions at the sampling sites being unsuitable for methanogenesis or the presence of more favorable electron acceptors in the groundwater. Consequently, the lack of the mcrA gene indicates an incomplete methanogenesis pathway, suggesting a limited risk of methane accumulation in the groundwater environment. And sulfur metabolism is dominated by assimilatory sulfate reduction, with cysD, cysH, and cysJ being the predominant genes, rather than dissimilatory sulfate reduction. These findings provide important evidence for the long-term performance and safety assessment of a geological disposal site for high-level radioactive waste in China.