In permafrost (perennially frozen ground) microbes survive oligotrophic conditions, sub-zero temperatures, low water availability and high salinity over millennia. Viable life exists in permafrost tens of thousands of years old but we know little about the metabolic and physiological adaptations to the challenges presented by life in frozen ground over geologic time. In this study we asked whether increasing age and the associated stressors drive adaptive changes in community composition and function. We conducted deep metagenomic and 16 S rRNA gene sequencing across a Pleistocene permafrost chronosequence from 19 000 to 33 000 years before present (kyr). We found that age markedly affected community composition and reduced diversity. Reconstruction of paleovegetation from metagenomic sequence suggests vegetation differences in the paleo record are not responsible for shifts in community composition and function. Rather, we observed shifts consistent with long-term survival strategies in extreme cryogenic environments. These include increased reliance on scavenging detrital biomass, horizontal gene transfer, chemotaxis, dormancy, environmental sensing and stress response. Our results identify traits that may enable survival in ancient cryoenvironments with no influx of energy or new materials.

Permafrost, characterized by its frozen soil, serves as a unique habitat for diverse microorganisms. Understanding these microbial communities is crucial for predicting the response of permafrost ecosystems to climate change. However, large-scale evidence regarding stratigraphic variations in microbial profiles remains limited. Here, we analyze microbial community structure and functional potential based on 16S rRNA gene amplicon sequencing and metagenomic data obtained from an ∼1000 km permafrost transect on the Tibetan Plateau. We find that microbial alpha diversity declines but beta diversity increases down the soil profile. Microbial assemblages are primarily governed by dispersal limitation and drift, with the importance of drift decreasing but that of dispersal limitation increasing with soil depth. Moreover, genes related to reduction reactions (e.g., ferric iron reduction, dissimilatory nitrate reduction, and denitrification) are enriched in the subsurface and permafrost layers. In addition, microbial groups involved in alternative electron accepting processes are more diverse and contribute highly to community-level metabolic profiles in the subsurface and permafrost layers, likely reflecting the lower redox potential and more complicated trophic strategies for microorganisms in deeper soils. Overall, these findings provide comprehensive insights into large-scale stratigraphic profiles of microbial community structure and functional potentials in permafrost regions.

The physical and chemical changes that accompany permafrost thaw directly influence the microbial communities that mediate the decomposition of formerly frozen organic matter, leading to uncertainty in permafrost–climate feedbacks. Although changes to microbial metabolism and community structure are documented following thaw, the generality of post‐thaw assembly patterns across permafrost soils of the world remains uncertain, limiting our ability to predict biogeochemistry and microbial community responses to climate change. Based on our review of the Arctic microbiome, permafrost microbiology, and community ecology, we propose that Assembly Theory provides a framework to better understand thaw‐mediated microbiome changes and the implications for community function and climate feedbacks. This framework posits that the prevalence of deterministic or stochastic processes indicates whether the community is well‐suited to thrive in changing environmental conditions. We predict that on a short timescale and following high‐disturbance thaw (e.g., thermokarst), stochasticity dominates post‐thaw microbiome assembly, suggesting that functional predictions will be aided by detailed information about the microbiome. At a longer timescale and lower‐intensity disturbance (e.g., active layer deepening), deterministic processes likely dominate, making environmental parameters sufficient for predicting function. We propose that the contribution of stochastic and deterministic processes to post‐thaw microbiome assembly depends on the characteristics of the thaw disturbance, as well as characteristics of the microbial community, such as the ecological and phylogenetic breadth of functional guilds, their functional redundancy, and biotic interactions. These propagate across space and time, potentially providing a means for predicting the microbial forcing of greenhouse gas feedbacks to global climate change.

Permafrost hosts a community of microorganisms that survive and reproduce for millennia despite extreme environmental conditions, such as water stress, subzero temperatures, high salinity, and low nutrient availability. Many studies focused on permafrost microbial community composition use DNA-based methods, such as metagenomics and 16S rRNA gene sequencing. However, these methods do not distinguish among active, dead, and dormant cells. This is of particular concern in ancient permafrost, where constant subzero temperatures preserve DNA from dead organisms and dormancy may be a common survival strategy. To circumvent this, we applied (i) LIVE/DEAD differential staining coupled with microscopy, (ii) endospore enrichment, and (iii) selective depletion of DNA from dead cells to permafrost microbial communities across a Pleistocene permafrost chronosequence (19,000, 27,000, and 33,000 years old). Cell counts and analysis of 16S rRNA gene amplicons from live, dead, and dormant cells revealed how communities differ between these pools, how they are influenced by soil physicochemical properties, and whether they change over geologic time. We found evidence that cells capable of forming endospores are not necessarily dormant and that members of the class Bacilli were more likely to form endospores in response to long-term stressors associated with permafrost environmental conditions than members of the Clostridia, which were more likely to persist as vegetative cells in our older samples. We also found that removing exogenous “relic” DNA preserved within permafrost did not significantly alter microbial community composition. These results link the live, dead, and dormant microbial communities to physicochemical characteristics and provide insights into the survival of microbial communities in ancient permafrost. IMPORTANCE Permafrost soils store more than half of Earth’s soil carbon despite covering ∼15% of the land area (C. Tarnocai et al., Global Biogeochem Cycles 23:GB2023, 2009, https://doi.org/10.1029/2008GB003327). This permafrost carbon is rapidly degraded following a thaw (E. A. G. Schuur et al., Nature 520:171–179, 2015, https://doi.org/10.1038/nature14338). Understanding microbial communities in permafrost will contribute to the knowledge base necessary to understand the rates and forms of permafrost C and N cycling postthaw. Permafrost is also an analog for frozen extraterrestrial environments, and evidence of viable organisms in ancient permafrost is of interest to those searching for potential life on distant worlds. If we can identify strategies microbial communities utilize to survive in permafrost, it may yield insights into how life (if it exists) survives in frozen environments outside of Earth. Our work is significant because it contributes to an understanding of how microbial life adapts and survives in the extreme environmental conditions in permafrost terrains.

Permanently frozen soil, or permafrost, covers a large portion of the Earth's terrestrial surface and represents a unique environment for cold-adapted microorganisms. As permafrost thaws, previously protected organic matter becomes available for microbial degradation. Microbes that decompose soil carbon produce carbon dioxide and other greenhouse gases, contributing substantially to climate change. Next-generation sequencing and other -omics technologies offer opportunities to discover the mechanisms by which microbial communities regulate the loss of carbon and the emission of greenhouse gases from thawing permafrost regions. Analysis of nucleic acids and proteins taken directly from permafrost-associated soils has provided new insights into microbial communities and their functions in Arctic environments that are increasingly impacted by climate change. In this article we review current information from various molecular -omics studies on permafrost microbial ecology and explore the relevance of these insights to our current understanding of the dynamics of permafrost loss due to climate change.