Anaerobic mineralization of 13C-labeled biomass by thermophilic microorganisms from aquifer sediments

High-Temperature Aquifer Thermal Energy Storage (HT-ATES) is a low-carbon energy system with high energy storage capacity, relying on the injection and extraction of heated (≥ 60℃) and cooled water in shallow aquifers. However, heat injection can alter aquifer microbial community composition, potentially affecting the degradation of dissolved organic matter (DOM) in groundwater. Therefore, understanding the structure, biodiversity, and physiology of thermophilic microorganisms in aquifer sediments—particularly their ability to degrade DOM—is essential for assessing the potential environmental impacts of HT-ATES systems. In this study, we tested whether 13 C-labeled microbial biomass is utilized by anaerobic thermophiles of pristine aquifer sediments at 60°C, 70°C, and 80°C. Biomass mineralization was observed in two replicates at 60°C and one at 70°C (out of six), but not at 80°C within 72 days. 16S rRNA gene sequencing revealed that bacterial diversity and richness at 60°C were significantly higher than at other temperatures. Distinct microbial communities developed in biomass-mineralizing and non-mineralizing cultures, with original sediment samples resembling 80°C incubations. In biomass-mineralizing cultures at 60°C and 70°C, phylotypes affiliated with spore-forming Bacillota developed. Brockia and Caldinitratiruptor dominated active replicates at 60°C, whereas Thermaerobacter was significantly abundant in the only active culture at 70°C. Our results show that some thermophilic, putative endospore-forming bacteria can utilize microbial biomass from natural environment as a carbon source at 60°C and 70°C, while the biomass was not utilized at 80°C. Therefore, during HT-ATES operations in pristine aquifers, thermophilic microbial communities may emerge and metabolize dead microbial biomass at temperatures up to 70°C. • Aquifer-derived thermophiles mineralized biomass anaerobically at 60°C and 70°C. • Mineralization was verified by 13 C-labelling of the microbial feed. • Temperature significantly affected microbial community composition.