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Despite being separated from the photosynthesis-driven surface by both distance and time, the deep biosphere is an important driver for the earth’s carbon and energy cycles. However, due to the difficulties in gaining access and low cell numbers, robust statistical omics studies have not been carried out, and this limits the conclusions that can be drawn. This study benchmarks the use of two separate sampling systems and demonstrates that they provide statistically similar RNA transcript profiles, importantly validating several previously published studies. The generated data are analyzed to identify statistically valid differences in active microbial community members and metabolic processes. The results highlight contrasting taxa and growth strategies in the modern marine waters that are influenced by recent infiltration of Baltic Sea water versus the hydrogen- and carbon dioxide-fed, extremely oligotrophic, thoroughly mixed water., Life in water-filled bedrock fissures in the continental deep biosphere is broadly constrained by energy and nutrient availability. Although these communities are alive, robust studies comparing active populations and metabolic processes across deep aquifers are lacking. This study analyzed three oligotrophic Fennoscandian Shield groundwaters, two “modern marine” waters that are replenished with organic carbon from the Baltic Sea and are likely less than 20 years old (171.3 and 415.4 m below sea level) and an extremely oligotrophic “thoroughly mixed” water (448.8 m below sea level) of unknown age that is composed of very old saline and marine waters. Cells were captured either using a sampling device that rapidly fixed RNA under in situ conditions or by filtering flowing groundwater over an extended period before fixation. Comparison of metatranscriptomes between the methods showed statistically similar transcript profiles for the respective water types, and they were analyzed as biological replicates. Study of the small subunit (SSU) rRNA confirmed active populations from all three domains of life, with many potentially novel unclassified populations present. Statistically supported differences between communities included heterotrophic sulfate-reducing bacteria in the modern marine water at 171.3 m below sea level that has a higher organic carbon content than do largely autotrophic populations in the H2- and CO2-fed thoroughly mixed water. While this modern marine water had signatures of methanogenesis, syntrophic populations were predominantly in the thoroughly mixed water. The study provides a first statistical evaluation of differences in the active microbial communities in groundwaters differentially fed by organic carbon or “geogases.”

A newly designed sampling apparatus was used to fix RNA under in situ conditions in the deep continental biosphere and benchmarks a strategy for deep biosphere metatranscriptomic sequencing. This apparatus enabled the identification of active community members and the processes they carry out in this extremely oligotrophic environment. This work presents for the first time evidence of eukaryotic, archaeal, and bacterial activity in two deep subsurface crystalline rock groundwaters from the Äspö Hard Rock Laboratory with different depths and geochemical characteristics. The findings highlight differences between organic carbon-fed shallow communities and carbon dioxide- and hydrogen-fed old saline waters. In addition, the data reveal a large portion of uncharacterized microorganisms, as well as the important role of candidate phyla in the deep biosphere, but also the disparity in microbial diversity when using standard microbial 16S rRNA gene amplification versus the large unknown portion of the community identified with unbiased metatranscriptomes., The continental subsurface is suggested to contain a significant part of the earth’s total biomass. However, due to the difficulty of sampling, the deep subsurface is still one of the least understood ecosystems. Therefore, microorganisms inhabiting this environment might profoundly influence the global nutrient and energy cycles. In this study, in situ fixed RNA transcripts from two deep continental groundwaters from the Äspö Hard Rock Laboratory (a Baltic Sea-influenced water with a residence time of

The continental deep biosphere is suggested to contain a substantial fraction of the earth's total biomass and microorganisms inhabiting this environment likely have a substantial impact on biogeochemical cycles. However, the deep microbial community is still largely unknown and can be influenced by parameters such as temperature, pressure, water residence times, and chemistry of the waters. In this study, 21 boreholes representing a range of deep continental groundwaters from the Äspö Hard Rock Laboratory were subjected to high-throughput 16S rRNA gene sequencing to characterize how the different water types influence the microbial communities. Geochemical parameters showed the stability of the waters and allowed their classification into three groups. These were (i) waters influenced by infiltration from the Baltic Sea with a "modern marine (MM)" signature, (ii) a "thoroughly mixed (TM)" water containing groundwaters of several origins, and (iii) deep "old saline (OS)" waters. Decreasing microbial cell numbers positively correlated with depth. In addition, there was a stronger positive correlation between increased cell numbers and dissolved organic carbon for the MM compared to the OS waters. This supported that the MM waters depend on organic carbon infiltration from the Baltic Sea while the ancient saline waters were fed by "geogases" such as carbon dioxide and hydrogen. The 16S rRNA gene relative abundance of the studied groundwaters revealed different microbial community compositions. Interestingly, the TM water showed the highest dissimilarity compared to the other two water types, potentially due to the several contrasting water types contributing to this groundwater. The main identified microbial phyla in the groundwaters were Gammaproteobacteria, unclassified sequences, Campylobacterota (formerly Epsilonproteobacteria), Patescibacteria, Deltaproteobacteria, and Alphaproteobacteria. Many of these taxa are suggested to mediate ferric iron and nitrate reduction, especially in the MM waters. This indicated that nitrate reduction may be a neglected but important process in the deep continental biosphere. In addition to the high number of unclassified sequences, almost 50% of the identified phyla were archaeal or bacterial candidate phyla. The percentage of unknown and candidate phyla increased with depth, pointing to the importance and necessity of further studies to characterize deep biosphere microbial populations.

The deep biosphere is the largest ‘bioreactor’ on earth, and microbes inhabiting this biome profoundly influence global nutrient and energy cycles. An important question for deep biosphere microbiology is whether or not specific populations are viable. To address this, we used quantitative PCR and high throughput 16S rRNA gene sequencing of total and viable cells (i.e. with an intact cellular membrane) from three groundwaters with different ages and chemical constituents. There were no statistically significant differences in 16S rRNA gene abundances and microbial diversity between total and viable communities. This suggests that populations were adapted to prevailing oligotrophic conditions and that non-viable cells are rapidly degraded and recycled into new biomass. With higher concentrations of organic carbon, the modern marine and undefined mixed waters hosted a community with a larger range of predicted growth strategies than the ultra-oligotrophic old saline water. These strategies included fermentative and potentially symbiotic lifestyles by candidate phyla that typically have streamlined genomes. In contrast, the old saline waters had more 16S rRNA gene sequences in previously cultured lineages able to oxidize hydrogen and fix carbon dioxide. This matches the paradigm of a hydrogen and carbon dioxide-fed chemolithoautotrophic deep biosphere., qPCR and 16S rRNA gene sequencing of total and viable cells (intact cellular membrane) from three groundwaters with different ages and chemical composition.