Identifying changes in the active, dead, and dormant microbial community structure across a chronosequence of ancient Alaskan permafrost

Permafrost— perennially frozen soil— hosts a diversity of microorganisms. Permafrost microbial communities 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, constant subzero temperatures may preserve DNA from dead organisms for extended periods of time making it difficult to distinguish between active, dead, and dormant cells. This is particularly concerning in increasingly ancient permafrost because dormancy may be a survival strategy and DNA from dead cells may accumulate over time. To circumvent this hurdle, we applied live/dead differential staining coupled with microscopy, endospore enrichment, and selective depletion of exogenous DNA and 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 spores 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 confirmed a previous study that permafrost age and percent ice content acted as drivers of microbial cell abundance and diversity. Total cell counts and alpha diversity decreased between our youngest and oldest samples, while the proportion of live cells increased in older samples compared to younger samples suggesting that the permafrost environment selects for organisms adapted to survive. Taken together, these data contribute to our understanding of how microbial life adapts and survives in the extreme permafrost environment across geologic timescales.