The subsurface biosphere in deep marine sediments is among the most challenging of microbial habitats; as energy sources become scarce and temperature increases with depth, microbial diversity declines and fewer microorganisms survive under these harsh conditions. However, this trend could potentially be attenuated by hydrothermal influence, since hydrothermal energy sources sustain microbial ecosystems with distinct chemolithotrophs (=microbes that use energy from the oxidation of inorganic compounds to sustain growth) and hyperthermophiles (=microbes that thrive in extremely hot environments). In this case, would chemolithotrophs and/or hyperthermophiles replace the mesophilic, heterotrophic communities that dominate benthic marine sediments, in particular when the geothermal heat gradient thins out the mesophiles with depth? Would these transitions be reflected in chemical parameters and sediment lithology? Such questions lead microbiologists and geochemists to study deep biosphere gradients with sequence-based methods that track uncultured and enigmatic bacteria and archaea that are approaching the limits of life, and to search for metabolic capabilities that change together with community composition and abundance.

In exploring these questions, we are returning to our favorite field site with well-characterized thermal and geochemical gradients. Guaymas Basin in the Gulf of California, Mexico, is a hydrothermally-influenced basin with massive 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 siliceous sediments that host abundant microbial populations towards increasingly baked, brittle, siliceous rocks and clays. At depth, sediments across the basin are intercalated with igneous intrusions of basaltic magma, called sills. Our study published in Communications Earth & Environment (Mara et al. 2024) leveraged deep subsurface sediment core samples obtained during International Ocean Discovery Program (IODP) Expedition 385, by drilling into Guaymas Basin’s subseafloor sediments and sill intrusions at eight sites with unique temperature and geochemical profiles (http://publications.iodp.org/proceedings/385 /385title.html). Perhaps no other marine region provides such a range of contrasting in-situ conditions that shape local microbial communities, including undisturbed benthic sediments, cold seeps and hydrothermal sites (Figure 1).

For two months during a typical expedition, drilling ship JOIDES Resolution hosts a diverse science crew of geologists, geophysicists, micropalaeontologists, chemists, and microbiologists, in addition to the drilling crew, housekeeping and galley staff, and scientific support technicians to oversee the core processing, shipboard core analyses, archival operations, and sample documentation. During this intense, immersive and multidisciplinary experience, scientists collect valuable deep subsurface samples (down to over 500 meters below the seafloor in Guaymas Basin) that could not be obtained in any other way; they also learn of each other’s research and build long-lasting collaborations that drive post-cruise research (Figure 2). For many seagoing scientists, a JOIDES Resolution expedition is heaven, admittedly of a peculiar and exhausting kind.

This particular study began after Guaymas Basin Expedition 385 and its participants returned to shore (Mara et al. 2024). Closely matching sediment samples for each collaborative team had been set aside for high-throughput sequencing of partial 16S rRNA genes from deep sediments using general prokaryotic primers that encompass bacteria and archaea, in conjunction with microscopic cell counts and analyses of mineral composition. It turned out that the Guaymas Basin subsurface hosts cosmopolitan subseafloor microbial communities dominated by largely uncultured Chloroflexota, Atribacteria and benthic archaea that are found in organic-rich continental margin sediments of all oceans. These are the heat-sensitive communities that flourish at cool and moderate temperatures near 20 °C, and whose numbers decline sharply at warm temperatures near 40 or 45 °C. Our previous metagenomic studies of this critical temperature range in the Guaymas Basin subsurface (Mara et al. 2023a) identified some hardy archaeal lineages within the Bathyarchaeia and Hadarchaeia that thrived specifically in deep, hot sediments near 50 and 60 °C, whereas bacteria disappeared from the metagenomic window at these depths and temperatures. To focus on archaea specifically, we undertook a second round of 16S rRNA gene analysis for all samples, this time based on archaea-specific primers that amplified a longer region of the 16S rRNA gene for improved phylogenetic resolution (approx. 800 base pairs compared to the 400 basepairs of the general prokaryotic survey). The archaeal community came into focus throughout the depth and temperature spectrum of all sampling sites; it turned out to be considerably more diverse, and represented more archaeal phylum-level lineages than the general primer survey had indicated. This time, we saw not only the common subsurface Bathyarchaeia (phylum Thermoproteota) and Marine Benthic Group D archaea (phylum Thermoplasmatota), but also Hadarchaota, Halobacterota (ANME-1 methane oxidizers), Euryarchaeota (Methanococci) and diverse lineages of the Asgardarchaea, now recognized as a superphylum.  

Which physical and geochemical factors are shaping the prokaryotic and archaeal datasets? Interestingly, the bacterially dominated prokaryotic amplicon dataset is influenced most consistently by porewater parameters linked to seawater penetration (sulfate and magnesium), organic matter content and remineralization (total organic carbon, ammonium), and sediment depth, whereas the archaeal amplicon profiles react to several alkali and transition metals (barium, mercury, rubidium), authigenic precipitates (dolomite) and carbon monoxide, in addition to total organic carbon and nitrogen, temperature and depth. In other words, the archaeal community seems to have forgotten any connection to the major seawater ions and instead relates to minerals or elements that accumulate at depth (dolomite), below the sulfate-methane transition zone (barium), or have hydrothermal or terrestrial sources (mercury). Interestingly, temperature appears as a major factor that structures the archaeal community, and indicates the possibility of a differentiated temperature response and thus temperature adapatations within the archaeal community – something that seems to get lost within the bacterially dominated prokaryotic dataset. It appears that bacteria are reaching for their smelling salts and collapse on the chaise longue when somebody mentions the T-word…. but the archaea are apparently not so delicate and deliver a visible response.

Without a good knowledge base that is anchored in microbial physiology and ecology,  taxonomic information based on 16S rRNA gene data cannot be linked specifically to many chemical factors; for example, it is impossible to tell which microbial group likes or dislikes rubidium, or to what degree this correlation reflects an actual causation that is rooted in microbial physiology. However, there is a knowledge base about temperature preferences, and we decided to search for archaeal thermophiles and hyperthermophiles based on the archaeal  16S rRNA gene dataset, taking advantage of the 800 basepair sequences for detailed phylogenetic placement and identification. Several thermophilic and hyperthermophilic archaeal lineages could be identified that either matched thermophilic enrichments (ANME-1 methane oxidizers), hydrothermal vent specialists with inferred thermophilic characteristics (Bathyarchaeial lineages), or cultured hyperthermophiles (Methanococcales). The latter constitute our best example for hyperthermophiles, since all Methanococcales-affiliated sequences in our dataset are close relatives of cultured, consistently hyperthermophilic and chemolithotrophic  (hydrogen-oxidizing) Methanocaldococcales species from hydrothermal vents. On the other hand, we did not know enough to identify specifically thermophilic candidate lineages among the Asgardarchaea and Hadarchaeota in Guaymas Basin. Tempting candidates that invite speculation are certainly available, for example a tantalizing lineage of Hadarchaeota from hot, deep aquifers about which – apart from the mere existence of the 16S rRNA gene sequences – nothing is known. The situation might improve in the future by thermophilic enrichments from hot springs, deep aquifers and hydrothermal vents.

Interestingly, the vast majority of archaeal thermophile and hyperthermophile sequences were found in sediments drilled near a major hydrothermal structure, called Ringvent, where a shallow, recently emplaced hot sill drives hydrothermal fluid circulation and creates very steep temperature gradients that reach  ca. 80 °C at the sediment/sill transition. Although drilling into the active hydrothermal crest of Ringvent was not possible for safety reasons, two drilling holes (U1547B and U1548B) were close enough to recover distinctly hydrothermal archaea that do not occur at other drilling locations. Interestingly, the subsurface sediments of Guaymas Basin reach temperatures similar to Ringvent (80 °C and up) in greater depths at other drilling sites, depending on the local heat flow and resulting temperature gradient; yet, these conditions do not result in the enrichment of hydrothermal hyperthermophiles. We conclude that our survey has detected a halo of hydrothermal archaea specifically around the actively venting Ringvent system.

Of course, this finding highlights the question whether non-hydrothermal thermophiles or hyperthermophiles are holding out in energy-depleted hot, deep sediments where hydrothermal carbon sources and electron couples are not available, and where circulation is impeded or entirely shut off by several hundred meters of overlying sediment. The previous metagenomic survey detected uncultured Hadarachaoeta and Bathyarchaeota specifically in these sediments at 50 to 60 °C (Mara et al. 2023a); alternatively, these archaea show up in the window of metagenomic detection when the mesophilic cosmopolitan sediment community has died off. These populations might be hiding in plain sight within the 16S rRNA gene dataset, where they remain incognito as long as metagenome and 16S rRNA gene data are not mutually linked and integrated. Clearly, the books on subsurface thermophiles remain open, and future progress in this field may require carefully disentangling hydrothermal vent thermophiles – the paradigmatic and relatively well-studied marine thermophiles – from other types of thermophiles that may have adapted to energy-depleted deep subsurface conditions, and that remain to be studied. Survival in the deep subsurface might imply long periods of extremely low activity or dormancy, during which transcriptional activity is down-regulated (Mara et al. 2023b) and critical biomolecules have to be maintained to preserve viability, even when nutrients and energy sources are limiting. Our transcriptome data indicate archaeal lineages that are actively engaged in this very challenging task.

For details, please see our manuscript: Deep subseafloor sediments in Guaymas Basin harbor cosmopolitan microbiota and traces of hydrothermal populations
DOI : 10.1038/s43247-024-01662-7
COMMSENV-24-0305

References

Mara, P., D. Beaudoin, I, Aiello, Y. Morono, D. Geller-McGrath, V.P. Edgcomb, A. Teske. 2024. Deep subseafloor sediments in Guaymas Basin harbor cosmopolitan microbiota and traces of hydrothermal populations. Communications Earth and Environment, 10.1038/s43247-024-01662-7.

Mara, P., D. Geller-McGrath, V. Edgcomb, D. Beaudoin, Y. Morono, A. Teske. 2023a. 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. 2023b. 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.

Contributors

Andreas Teske¹, Paraskevi Mara², David Beaudoin³, Yuki Morono⁴, Ivano Aiello⁵, Virginia P. Edgcomb²

¹ Department of Earth, Marine and Environmental Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA

² Geology and Geophysics Department, Woods Hole Oceanographic Institution, Woods Hole, MA, 02543, USA

³ Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA, 02543, USA

⁴ Kochi Institute for Core Sample Research, Institute for Extra-cutting-edge Science and Technology Avantgarde Research (X-STAR), Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Monobe, Nankoku, Kochi, Japan

⁵ San Jose State University, Moss Landing Marine Laboratory, Moss Landing, CA 95039, USA