Changes in the active, dead, and dormant microbial community structure across a Pleistocene permafrost chronosequence

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 metagenomic and 16S rRNA gene sequencing. However, these methods do not distinguish between 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 thousand years (K), 27K, and 33K). 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 class Bacilli were more likely to form endospores in response to long-term stressors associated with permafrost environmental conditions than members of 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 provides insights into the survival of microbial communities in ancient permafrost.