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

Abstract

29Permafrost hosts a community of microorganisms that survive and reproduce for 30 millennia despite extreme environmental conditions such as water stress, subzero 31 temperatures, high salinity, and low nutrient availability. Many studies focused on 32 permafrost microbial community composition use DNA-based methods such as 33 metagenomic and 16S rRNA gene sequencing. However, these methods do not 34 distinguish between active, dead, and dormant cells. This is of particular concern in 35 ancient permafrost where constant subzero temperatures preserve DNA from dead 36 organisms and dormancy may be a common survival strategy. To circumvent this 37 we applied: (i) live/dead differential staining coupled with microscopy, (ii) 38 endospore enrichment, and (iii) selective depletion of DNA from dead cells to 39 permafrost microbial communities across a Pleistocene permafrost chronosequence 40 (19K, 27K, and 33K). Cell counts and analysis of 16S rRNA gene amplicons from 41 live, dead, and dormant cells revealed how communities differ between these pools 42 and how they change over geologic time. We found clear evidence that cells capable 43 of forming endospores are not necessarily dormant and that the propensity to form 44 endospores differed among taxa. Specifically, Bacilli are more likely to form 45 endospores in response to long-term stressors associated with permafrost 46 environmental conditions than members of Clostridia, which are more likely to 47 content largely control microbial community diversity and cell abundances. 52 53 IMPORTANCE 54 The study of permafrost transcends the study of climate change and 55 exobiology. Permafrost soils store more than half earth's soil carbon despite 56 covering ~15% of the land area (Tarnocai et al 2009). This permafrost carbon is 57 rapidly degraded following thaw (Tarnocai C et al 2009, Schuur et al 2015) . 58 Understanding microbial communities in permafrost will contribute to the 59 knowledge base necessary to understand the rates and forms of permafrost C and N 60 cycling post thaw. Permafrost is also an analog for frozen extraterrestrial 61 environments and evidence of viable organisms in ancient permafrost is of interest 62 to those searching for potential life on distant worlds. If we can identify strategies 63 microbial communities utilize to survive permafrost we can focus efforts searching 64 for evidence of life on cryogenic cosmic bodies. Our work is significant because it 65 contributes to an understanding of how microbial life adapts and survives in the 66 extreme environmental conditions in permafrost terrains across geologic timescales. 67 68 drastically alter estimates of diversity and abundance. Furthermore, because many 75 communities host dormant cells, DNA-based approaches may not represent active 76 members. In temperate soils, up to 40% of DNA is from dead or compromised cells (3). 77In permafrost, the amount of 'relic' DNA may be even higher because frozen conditions 78 preserve DNA from dead cells. In non-permafrost environments, multi-omic approac...

Life at the Frozen Limit: Microbial Carbon Metabolism Across a Late Pleistocene Permafrost Chronosequence

“…Yet the variation in OM chemistry is not well constrained, in part because of our limited understanding of the initial OM entrained in permafrost, but also because of poor recognition that microbial communities in permafrost are themselves altering its chemical nature. Recently, Mackelprang et al demonstrated that microbial populations adapt and survive in permafrost across the Fox Tunnel chronosequence ( Mackelprang et al, 2017 ; Burkert et al, 2019 ). These previous studies of the Fox Permafrost Tunnel found that the relative abundance of genes and pathways related to long-term survival increased with age and that vegetative cells persist across the same chronosequence ( Mackelprang et al, 2017 ; Burkert et al, 2019 ).…”

“…Recently, Mackelprang et al demonstrated that microbial populations adapt and survive in permafrost across the Fox Tunnel chronosequence ( Mackelprang et al, 2017 ; Burkert et al, 2019 ). These previous studies of the Fox Permafrost Tunnel found that the relative abundance of genes and pathways related to long-term survival increased with age and that vegetative cells persist across the same chronosequence ( Mackelprang et al, 2017 ; Burkert et al, 2019 ). However, the question of how these microbial populations interact with permafrost OC remains.…”

“…Permafrost soils host a diverse microbial community, though diversity is typically reduced relative to active layer soils, pointing to its unique environmental stressors (e.g., reduction in energy availability and salinity) ( Mackelprang et al, 2011 ; Hultman et al, 2015 ; Rivkina et al, 2016 ). We still do not understand the mechanisms that enable microbial survival and growth in permafrost nor how microbial communities respond to increasingly thermodynamically limited conditions given the closed nature of the system ( Rivkina et al, 2000 ; Tuorto et al, 2014 ; Hultman et al, 2015 ; Burkert et al, 2019 ). The overall understanding of C cycling-related metabolic processes that occur below 0°C may be inferred from genetic changes in the microbial community and the distribution of functional traits ( Mackelprang et al, 2011 ; Hultman et al, 2015 ).…”

“…The overall understanding of C cycling-related metabolic processes that occur below 0°C may be inferred from genetic changes in the microbial community and the distribution of functional traits ( Mackelprang et al, 2011 ; Hultman et al, 2015 ). By linking DOC quality and molecular composition of intact permafrost OM with microbial C-processing genes it may be possible to reconstruct the pathways used for microbial OM metabolism which enable survival across millennia or longer ( Tveit et al, 2013 , 2015 ; Bottos et al, 2018 ; Burkert et al, 2019 ; Liang et al, 2019 ).…”

Microbial genomics amidst the Arctic crisis

“…Recent chronosequence surveys [ 63, 64 ] have examined the survival mechanisms of viable microbiota within permafrost dating from the Pleistocene period (samples that dated to 19 000–33 000 years before the present). These reveal that while endospore-forming taxa are prevalent, viable biomass from some of these taxa remain as vegetative cells [ 64 ], underlining the potential for long-term, low-growth-rate survival rather than sporulation as a persistence mechanism within permafrost.…”