Changes in the Active, Dead, and Dormant Microbial Community Structure Across a Pleistocene Permafrost Chronosequence

Permafrost hosts a community of microorganisms, which survive and reproduce for millennia despite extreme conditions such as water stress, subzero temperatures, high salinity, and low nutrient availability. Most studies targeting permafrost microbial communities use DNA-based methods such as metagenomic and 16S rRNA gene sequencing. However, these methods do not distinguish between active, dead, and dormant cells. This is particularly concerning in ancient permafrost where constant subzero temperatures preserve DNA from dead organisms and dormancy may be a common survival strategy. To circumvent this hurdle, 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 kyr, 27 kyr, and 33 kyr). Cell counts and analysis of 16S rRNA gene amplicons from live, dead, and dormant cells revealed how communities differ between these pools and how they change over geologic time. We found clear evidence that cells capable of forming endospores are not necessarily dormant and that the propensity to form endospores differed between taxa. Specifically, Bacilli are more likely to form endospores in response to long-term stressors associated with life in permafrost than members of Clostridia, which are more likely to persist as vegetative cells over geologic timescales. We also found that exogenous DNA preserved within permafrost does not bias DNA sequencing results, since its removal did not significantly alter microbial community composition. Lastly, the results of our cell enumeration extended results from a previous study showing that permafrost age and ice content drove microbial community diversity and cell abundances.