The Eger Rift subsurface is characterized by frequent seismic activity and consistently high CO2 concentrations, making it a unique deep biosphere ecosystem and a suitable site to study the interactions between volcanism, tectonics, and microbiological activity. Pulses of geogenic H2 during earthquakes may provide substrates for methanogenic and chemolithotrophic processes, but very little is currently known about the role of subsurface microorganisms and their cellular processes in this type of environment. To assess the impact of geologic activity on microbial life, we analyzed the geological, geochemical, and microbiological composition of rock and sediment samples from a 240 m deep drill core, running across six lithostratigraphic zones. In addition, we evaluated diversity as well as metabolic attributes of bacterial and archaeal communities. Our investigation revealed a distinct low biomass community, with a surprisingly diverse Archaea population, providing strong support that methanogenic archaea reside in the Eger subsurface. Geochemical analysis revealed sulfate and sodium concentrations as high as 1000 mg L-1 in sediment samples from a depth between 50 and 100 m and in weathered rock samples collected below 200 m. Most microbial signatures could be assigned to common soil and water bacteria, which together with the occurrence of freshwater Cyanobacteria at specific depths, emphasize the heterogenous, groundwater movement driven nature of this terrestrial subsurface environment. Although not as frequently and abundantly as initially expected, our investigations also found evidence for anaerobic, autotrophic, and acidophilic communities in Eger Rift sediments, as sulfur cycling taxa like Thiohalophilus and Desulfosporosinus were specifically enriched at depths below 100 m. The detection of methanogenic, halophilic, and ammonia oxidizing archaeal populations demonstrate that the unique features of the Eger Rift subsurface environment provide the foundation for diverse types of microbial life, including the microbial utilization of geologically derived CO2 and when available H2, as a primary energy source.

A metagenome-assembled genome (MAG), named Methanosarcina sp. strain ERenArc_MAG2, was obtained from a 3-month-old H 2 /CO 2 atmosphere enrichment culture, originally inoculated with 60-m deep drill core sediment collected from the tectonic Eger Rift terrestrial subsurface. Annotation of the recovered draft genome revealed putative archaeal methanogenesis genes in the deep biosphere.

<p>The Eger Rift (Czech Republic) is characterized by deep-seated volcanic activity, leading to high CO<sub>2</sub> fluxes up to 125 kg m<sup>-2</sup> d<sup>-1</sup> and frequent tectonic activity. With its subsurface being naturally CO<sub>2</sub>-rich at least since the Mid-Pleistocene, the Eger Rift is a natural analogue of underground CO<sub>2</sub> storage sites that allows for studying long-term effects of high CO<sub>2</sub> concentrations on the mineralogy and microbiology of such systems. Frequent small earthquakes lead to abiotic production of H<sub>2</sub>, 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 CO<sub>2</sub> storage. To assess the metabolic potential of the CO<sub>2</sub>-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 CO<sub>2</sub> conditions, and their changes after exposure to Hydrogen.</p> <p>A 230 m long drill core was recovered as part of the International Continental Drilling Program’s (ICDP) Eger Rift Project. Drilling was carried out under contamination-controlled conditions to provide pristine samples for geomicrobiological analyses.</p> <p>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 CO<sub>2</sub>-adapted microbial communities to utilize Hydrogen. We further isolated and purified active methanogens for detailed insights into their metabolic capability.</p> <p>Our investigation revealed a CO<sub>2</sub>-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 <em>Methanobacterium</em> and <em>Methanosphaerula. </em><em>T</em>he autotrophic sulfate reducer <em>Desulfosporosinus,</em> 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.</p> <p>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.</p>

A circular, single-contig Methanobacterium sp. metagenome-assembled genome (MAG) was recovered from high-CO 2 enrichments inoculated with drill core material from the tectonic Eger Rift terrestrial subsurface. Annotation of the recovered MAG highlighted putative methanogenesis genes, providing valuable information on archaeal activity in the deep biosphere.

IntroductionLong-term stability of underground CO2 storage is partially affected by microbial activity but our knowledge of these effects is limited, mainly due to a lack of sites. A consistently high flux of mantle-derived CO2 makes the Eger Rift in the Czech Republic a natural analogue to underground CO2 storage. The Eger Rift is a seismically active region and H2 is produced abiotically during earthquakes, providing energy to indigenous microbial communities.MethodsTo investigate the response of a microbial ecosystem to high levels of CO2 and H2, we enriched microorganisms from samples from a 239.5 m long drill core from the Eger Rift. Microbial abundance, diversity and community structure were assessed using qPCR and 16S rRNA gene sequencing. Enrichment cultures were set up with minimal mineral media and H2/CO2 headspace to simulate a seismically active period with elevated H2.Results and discussionMethane headspace concentrations in the enrichments indicated that active methanogens were almost exclusively restricted to enrichment cultures from Miocene lacustrine deposits (50–60 m), for which we observed the most significant growth. Taxonomic assessment showed microbial communities in these enrichments to be less diverse than those with little or no growth. Active enrichments were especially abundant in methanogens of the taxa Methanobacterium and Methanosphaerula. Concurrent to the emergence of methanogenic archaea, we also observed sulfate reducers with the metabolic ability to utilize H2 and CO2, specifically the genus Desulfosporosinus, which were able to outcompete methanogens in several enrichments. Low microbial abundance and a diverse non-CO2 driven microbial community, similar to that in drill core samples, also reflect the inactivity in these cultures. Significant growth of sulfate reducing and methanogenic microbial taxa, which make up only a small fraction of the total microbial community, emphasize the need to account for rare biosphere taxa when assessing the metabolic potential of microbial subsurface populations. The observation that CO2 and H2-utilizing microorganisms could only be enriched from a narrow depth interval suggests that factors such as sediment heterogeneity may also be important. This study provides new insight on subsurface microbes under the influence of high CO2 concentrations, similar to those found in CCS sites.