Frozen in Time? Microbial Strategies for Survival and Carbon Metabolism over Geologic Time in a Pleistocene Permafrost Chronosequence.

Permafrost soils have received tremendous interest due to their importance as a global carbon store with the potential to be thawed over the coming centuries. Instead of being ‘frozen in time,’ permafrost contains active microorganisms.  Most metagenomic studies have focused on Holocene aged permafrost. Here, we target Pleistocene aged ice and carbon rich permafrost (Yedoma), which can differ in carbon content and stage of decay. Our aim was to understand how microbes in the permafrost transform organic matter over geologic time and to identify physiological and biochemical adaptations that enable long-term survival. We used next-generation sequencing to characterize microbial communities along a permafrost age gradient. Samples were collected from the Cold Regions Research and Engineering Laboratory (CRREL) Permafrost Tunnel near Fox, AK, which penetrates a hillside providing access to permafrost ranging in age from 12 to 40 kypb.  DNA was extracted directly from unthawed samples. 16S rRNA amplicon (16S) and shotgun metagenome sequencing revealed significant age-driven differences.  First, microbial diversity declines with permafrost age, likely due to long-term exposure to environmental stresses and a reduction in metabolic resources.  Second, we observed taxonomic differences among ages, with an increasing abundance of Firmicutes (endospore-formers) in older samples, suggesting that dormancy is a common survival strategy in older permafrost.  Ordination of 16S and metagenome data revealed age-based clustering. Genes differing significantly between age categories included those involved in lipopolysaccharide assembly, cold-response, and carbon processing.  These data point to the physiological adaptations to long-term frozen conditions and to the metabolic processes utilized in ancient permafrost. In fact, a gene common in older samples is involved in cadaverine production, which could potentially explain the putrefied smell of Pleistocene aged permafrost. Coupled with soil chemistry analysis, these processes show how a tightly linked microbial food web can survive over geologic time with no influx of new energy or materials.  This web may also help to explain differences in Pleistocene carbon chemistry and why this carbon is highly bioavailable for microbial consumption post thaw.