The subsurface biosphere in deep sediments and rocks is among the most challenging of microbial habitats; as temperature increases downwards and energy sources become scarce, fewer and fewer microorganisms manage to survive. Will bacteria and archaea gradually decline in cell numbers and diversity, and ultimately disappear one after another as they can no longer cope with intensifying subsurface stress? Or do deep subsurface habitats select for particularly hardy microbes that thrive or at least survive under conditions where few others can? These questions lead microbiologists and geochemists to study deep biosphere gradients with methods that include metagenomics to track uncultured and enigmatic bacteria and archaea that are approaching the limits of life, and to obtain insights into metabolic capabilities that may determine survival or die-off under extreme conditions.

            Such studies require a field site with well-characterized thermal and geochemical gradients. Guaymas Basin in the Gulf of California, Mexico, is a hydrothermally-influenced basin with thick (100’s of meters) organic-rich sediments punctuated in different locations by hydrothermal hot spots and hydrocarbon seeps that form a complex benthic landscape. Under the influence of increasing depth and temperature, sediments change from relatively soft, predominantly biogenic sediments with abundant microbial populations towards increasingly baked, hardened cherts and clays. At depth, sediments across the basin are intercalated with volcanic basalt intrusions, called sills. Sampling these subsurface sediments and volcanic sills requires a specialized deep-sea drill ship: D/V JOIDES Resolution  carries a large drilling crew, fully equipped shipboard laboratories and up to 30 scientists to perform scientific ocean drilling around the globe, run by the International Ocean Discovery Program (IODP) (Figure 1).

Our study published in Nature Communications (Mara et al. 2023) leveraged deep subsurface sediment core samples obtained during IODP Expedition 385, by drilling into Guaymas Basin’s hydrothermally influenced seafloor sediments and sill intrusions at eight sites with unique temperature and geochemical profiles (http://publications.iodp.org/proceedings/ 385/385title.html). Perhaps no other location provides such a range of contrasting in-situ conditions that shape local microbial communities. Microbiologists, biochemists, geologists, geophysicists, micropaleontologists and chemists spent 8 weeks together on the D/V JOIDES Resolution in Guaymas Basin. The JOIDES Resolution operates much like an autonomous city, carrying not only the ship’s crew, but a drilling crew, a food and hotel crew, and a scientific support team from Texas A&M University to oversee the core processing, shipboard core analyses, archival operations, and detailed sample documentation procedures. During this intense, immersive and multidisciplinary experience, scientists not only collect highly valuable samples, but they learn of each other’s research and build lasting collaborations that drive post-cruise research (Figure 2).

By analyzing metagenome-assembled genomes (MAGs) from deep sediments in tandem with their in-situ temperatures and geochemical conditions, it became clear that chemical parameters influenced the composition of bacterial and archaeal MAGs at cool and intermediate temperatures, but temperature in itself was the most important variable in determining MAG composition above ca. 45 °C. Microbial diversity, and average genome size of the microbial survivors, decreased quickly with depth and temperature. While widely distributed subsurface bacteria (Chloroflexota, Acidobacterota, Desulfobacterota) dominated in cool and temperate sediments and gradually declined with increasing temperature and depth, we found that family-level lineages within the Bathyarchaeia and Hadarchaeia thrived specifically in deep, hot sediments near 50 and 60 °C, and bucked the trend towards downcore microbial extinction. These resilient subsurface archaea appear to constitute a deep, hot biosphere that is distinct from the subsurface microbiota of cold sediments, and from hyperthermophiles that dominate in near-surface, energy-rich sediments at hydrothermal vents.

            How do bacteria and archaea survive and thrive in the deep subsurface? The genomes of detected taxa reflected metabolisms that were predicted for the deep biosphere, including sulfur, metal and methane cycling, hydrocarbon degradation, and carbon fixation. Metatranscriptome analyses of Guaymas Basin deep subsurface samples (Mara et al. 2023) indicate that microorganisms continue to take the first step toward metabolic activity by transcribing genes from a wide spectrum of pathways. While this indicates to us that there are living cells in the Guaymas Basin deep biosphere, given the nutritional and energetic limitations of these habitats, it challenges our ideas of what it means to be alive. In addition to predicted metabolisms, and of particular interest to us, were genomic features that can have adaptive value for the deep biosphere where carbon and energy sources as well as sources of electron acceptors are more limited with depth below seafloor. According to the metatranscriptome results, cells appear to be investing most heavily in expression of genes associated with DNA and RNA repair and stability, RNA modifications, and membrane repair, not so much in expression of genes for sulfur, metal and methane cycling, hydrocarbon degradation, and carbon fixation. While it appears that there are some cells actively engaging in those metabolisms, it seems likely that the vast majority of cells do not have access to adequate carbon, energy, or electron acceptors for active growth or division. Instead, cells appear to be somewhere along the continuum between dead, dormant, or just barely active, enough only to control entropy until conditions improve, perhaps waiting over geological timeframes without cell division or genome replication. Interestingly, many inhabitants of the Guaymas deep biosphere contain genes for two-component systems that can induce metabolic shifts in response to environmental change, transporters and efflux pumps associated with microbial defense, and biosynthetic gene clusters involved in synthesis of diverse secondary metabolites. However, unless some environmental signal triggers the expression of these genes, a subsurface community may largely persist in a state of suspended animation.

Clearly, special adaptations are required for cells to survive potentially long periods of extremely low activity or dormancy, during which stability of critical biomolecules must be ensured in order for other activities to resume once conditions are more favorable. Life in such a “slow lane” implies that evolution in the deep biosphere must consequently proceed at extremely slow rates. Life in the slow lane also imposes limits on thermophilic life, since damage to cellular components cannot be repaired quickly when nutrients and energy sources are limiting. Thus, the survival tolerance of microbial life should not be measured against isolated physical and chemical parameters, but by their naturally existing combination; tracing these limits may provide valuable markers for life’s range and boundaries.

References

Mara, P., D. Geller-McGrath, V. Edgcomb, D. Beaudoin, Y. Morono, A. Teske. 2023. Metagenomic Profiles of Archaea and Bacteria within Thermal and Geochemical Gradients of the Guaymas Basin Deep Subsurface. Nature Communications 14:7768, doi: 10.1038/s41467-023-43296-x.

Mara, P., Zhou, Y., Teske, A., Morono, Y., Beaudoin, D., Edgcomb, V.P. 2023. Microbial gene expression in Guaymas Basin subsurface sediments responds to hydrothermal stress and energy limitation. The ISME Journal 17:1907-1919, doi: 10.1038/s41396-023-01492-z.

Blog by Andreas Teske, Yuki Morono, and Virginia Edgcomb