Rare methanogenic Archaea can become active in natural high-CO2 subsurface environments upon changing environmental conditions
Authors: Jia Zeyu , Lipus Daniel , Burckhardt Oliver , Bussert Robert , Sondermann Megan , Bartholomaeus Alexander , Wagner Dirk , Kallmeyer Jens 
Published: February 2023

The Eger Rift (Czech Republic) is characterized by deep-seated volcanic activity, leading to high CO2 fluxes up to 125 kg m-2 d-1 and frequent tectonic activity. With its subsurface being naturally CO2-rich at least since the Mid-Pleistocene, the Eger Rift is a natural analogue of underground CO2 storage sites that allows for studying long-term effects of high CO2 concentrations on the mineralogy and microbiology of such systems. Frequent small earthquakes lead to abiotic production of H2, providing energy to indigenous microbial communities. Investigating the microbial communities residing in such natural analogue sites provides crucial knowledge about the possible log-term consequences of anthropogenic underground CO2 storage. To assess the metabolic potential of the CO2-adapted microbial community and its reaction to transient availability of Hydrogen, we evaluated diversity as well as metabolic attributes of bacterial and archaeal communities surviving under high CO2 conditions, and their changes after exposure to Hydrogen.A 230 m long drill core was recovered as part of the International Continental Drilling Program&#8217;s (ICDP) Eger Rift Project. Drilling was carried out under contamination-controlled conditions to provide pristine samples for geomicrobiological analyses.We used cell counts and qPCR to assess microbial abundance across sediment and rock samples and both Illumina and Nanopore DNA sequencing platforms to gain insights into community structure and metabolic potential. Enrichments were set up to evaluate the ability of the CO2-adapted microbial communities to utilize Hydrogen. We further isolated and purified active methanogens for detailed insights into their metabolic capability.Our investigation revealed a CO2-adapted community with low biomass and a surprisingly diverse archaeal population. Methanogens are rare and account for less than 1% of the total microbial community in most drill core samples. However, enrichments revealed an active hydrogenotrophic methanogen population from a narrow depth interval (50-60 m), dominated by Methanobacterium and Methanosphaerula. The autotrophic sulfate reducer Desulfosporosinus, also thrives in the same depth interval. We isolated methanogen strains from the enrichments from the 50-60 m depth interval, whereas enrichments from other depths remained low in biomass and showed little or no methanogenesis.The strong differences in methanogenic activity among the enrichment cultures emphasize sediment heterogeneity, strongly suggesting the need for a high-resolution sampling strategy to evaluate the long-term effects of CCS. Our study shows that distinct processes may happen only in very narrow depth intervals and only reveal themselves through incubation/cultivation experiments, thus highlighting the importance of cultivation-dependent investigation on exploring the metabolic potential of microbial communities in subsurface environments.