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Biofilm formation is a common adaptation for microbes in energy-limited conditions such as those prevalent in the vast deep terrestrial biosphere. However, due to the low biomass and the inaccessible nature of subsurface groundwaters, the microbial populations and genes involved in its formation are understudied. Here, a flow-cell system was designed to investigate biofilm formation under in situ conditions in two groundwaters of contrasting age and geochemistry at the Äspö Hard Rock Laboratory, Sweden. Metatranscriptomes showed Thiobacillus, Sideroxydans, and Desulforegula to be abundant and together accounted for 31% of the transcripts in the biofilm communities. Differential expression analysis highlighted Thiobacillus to have a principal role in biofilm formation in these oligotrophic groundwaters by being involved in relevant processes such as the formation of extracellular matrix, quorum sensing, and cell motility. The findings revealed an active biofilm community with sulfur cycling as a prominent mode of energy conservation in the deep biosphere., (© 2023. The Author(s).)

While oligotrophic deep groundwaters host active microbes attuned to the low-end of the bioenergetics spectrum, the ecological constraints on microbial niches in these ecosystems and their consequences for microbiome convergence are unknown. Here, we provide a genome-resolved, integrated omics analysis comparing archaeal and bacterial communities in disconnected fracture fluids of the Fennoscandian Shield in Europe. Leveraging a dataset that combines metagenomes, single cell genomes, and metatranscriptomes, we show that groundwaters flowing in similar lithologies offer fixed niches that are occupied by a common core microbiome. Functional expression analysis highlights that these deep groundwater ecosystems foster diverse, yet cooperative communities adapted to this setting. We suggest that these communities stimulate cooperation by expression of functions related to ecological traits, such as aggregate or biofilm formation, while alleviating the burden on microorganisms producing compounds or functions that provide a collective benefit by facilitating reciprocal promiscuous metabolic partnerships with other members of the community. We hypothesize that an episodic lifestyle enabled by reversible bacteriostatic functions ensures the subsistence of the oligotrophic deep groundwater microbiome., (© 2021. The Author(s).)

The deep biosphere contains members from all three domains of life along with viruses. Here we investigate the deep terrestrial virosphere by sequencing community nucleic acids from three groundwaters of contrasting chemistries, origins, and ages. These viromes constitute a highly unique community compared to other environmental viromes and sequenced viral isolates. Viral host prediction suggests that many of the viruses are associated with Firmicutes and Patescibacteria, a superphylum lacking previously described active viruses. RNA transcript-based activity implies viral predation in the shallower marine water-fed groundwater, while the deeper and more oligotrophic waters appear to be in 'metabolic standby'. Viral encoded antibiotic production and resistance systems suggest competition and antagonistic interactions. The data demonstrate a viral community with a wide range of predicted hosts that mediates nutrient recycling to support a higher microbial turnover than previously anticipated. This suggests the presence of 'kill-the-winner' oscillations creating slow motion 'boom and burst' cycles.

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 H 2 - and CO 2 -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." IMPORTANCE 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., (Copyright © 2019 Lopez-Fernandez et al.)

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 <20 years, defined as "modern marine," and an "old saline" groundwater with a residence time of thousands of years) were subjected to metatranscriptome sequencing. Although small subunit (SSU) rRNA gene and mRNA transcripts aligned to all three domains of life, supporting activity within these community subsets, the data also suggested that the groundwaters were dominated by bacteria. Many of the SSU rRNA transcripts grouped within newly described candidate phyla or could not be mapped to known branches on the tree of life, suggesting that a large portion of the active biota in the deep biosphere remains unexplored. Despite the extremely oligotrophic conditions, mRNA transcripts revealed a diverse range of metabolic strategies that were carried out by multiple taxa in the modern marine water that is fed by organic carbon from the surface. In contrast, the carbon dioxide- and hydrogen-fed old saline water with a residence time of thousands of years predominantly showed the potential to carry out translation. This suggested these cells were active, but waiting until an energy source episodically becomes available. IMPORTANCE 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., (Copyright © 2018 Lopez-Fernandez et al.)

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.