Disconnected active layers and unfrozen permafrost: A discussion of permafrost-related terms and definitions.

Permafrost is ground that remains at or below 0 °C for two or more consecutive years. It is overlain by an active layer which thaws and freezes annually. The difference between these definitions - the active layer based on pore water phase and permafrost based on soil temperature - leads to challenges when monitoring and modelling permafrost environments. Contrary to its definition, the key properties of permafrost including hardness, bearing capacity, permeability, unfrozen water content, and energy content, depend primarily on the ice content of permafrost and not its temperature. Temperature-based measurements in permafrost systems often overlook key features, e.g. taliks and cryopegs, and comparisons between measured and modelled systems can differ energetically by up to 90 % while reporting the same temperature. Due to the shortcomings of the temperature-based definition, it is recommended that an estimate of ice content be reported alongside temperature in permafrost systems for both in-situ measurements and modelling applications. PLAIN LANGUAGE SUMMARY: Permafrost is ground that remains at or below 0 °C for two or more consecutive years. Above it sits an active layer which thaws and freezes annually (meaning that the water in the ground changes to ice each winter). The difference between these definitions - the active layer based on the state or water in the ground and permafrost based on ground temperature - leads to challenges when measuring (in the field) and modelling (using computers) permafrost environments. In addition to these challenges, the key properties of permafrost including its ability to support infrastructure, convey water, and absorb energy depend more on its ice content than its temperature. Due to the shortcomings of the temperature-based definition, it is recommended that an estimate of ice content be reported alongside temperature in permafrost systems for both field measurements and modelling applications.

Microbial Diversity and Function in Shallow Subsurface Sediment and Oceanic Lithosphere of the Atlantis Massif.

The marine lithospheric subsurface is one of the largest biospheres on Earth; however, little is known about the identity and ecological function of microorganisms found in low abundance in this habitat, though these organisms impact global-scale biogeochemical cycling. Here, we describe the diversity and metabolic potential of sediment and endolithic (within rock) microbial communities found in ultrasmall amounts (101 to 104 cells cm-3) in the subsurface of the Atlantis Massif, an oceanic core complex on the Mid-Atlantic Ridge that was sampled on International Ocean Discovery Program (IODP) Expedition 357. This study used fluorescence-activated cell sorting (FACS) to enable the first amplicon, metagenomic, and single-cell genomic study of the shallow (<20 m below seafloor) subsurface of an actively serpentinizing marine system. The shallow subsurface biosphere of the Atlantis Massif was found to be distinct from communities observed in the nearby Lost City alkaline hydrothermal fluids and chimneys, yet similar to other low-temperature, aerobic subsurface settings. Genes associated with autotrophy were rare, although heterotrophy and aerobic carbon monoxide and formate cycling metabolisms were identified. Overall, this study reveals that the shallow subsurface of an oceanic core complex hosts a biosphere that is not fueled by active serpentinization reactions and by-products. IMPORTANCE The subsurface rock beneath the ocean is one of the largest biospheres on Earth, and microorganisms within influence global-scale nutrient cycles. This biosphere is difficult to study, in part due to the low concentrations of microorganisms that inhabit the vast volume of the marine lithosphere. In spite of the global significance of this biosphere, little is currently known about the microbial ecology of such rock-associated microorganisms. This study describes the identity and genomic potential of microorganisms in the subsurface rock and sediment at the Atlantis Massif, an underwater mountain near the Mid-Atlantic Ridge. To enable our analyses, fluorescence-activated cell sorting (FACS) was used as a means to concentrate cells from low biomass environmental samples for genomic analyses. We found distinct rock-associated microorganisms and found that the capacity for microorganisms to utilize organic carbon was the most prevalent form of carbon cycling. We additionally identified a potential role for carbon monoxide metabolism in the subsurface.

Variability in carbon uptake and (re)cycling in Antarctic cryptoendolithic microbial ecosystems demonstrated through radiocarbon analysis of organic biomarkers.

Cryptoendolithic lichens and cyanobacteria living in porous sandstone in the high-elevation McMurdo Dry Valleys are purported to be among the slowest growing organisms on Earth with cycles of death and regrowth on the order of 103 -104  years. Here, organic biomarker and radiocarbon analysis were used to better constrain ages and carbon sources of cryptoendoliths in University Valley (UV; 1,800 m.a.s.l) and neighboring Farnell Valley (FV; 1,700 m.a.s.l). Δ14 C was measured for membrane component phospholipid fatty acids (PLFA) and glycolipid fatty acids, as well as for total organic carbon (TOC). PLFA concentrations indicated viable cells comprised a minor (<0.5%) component of TOC. TOC Δ14 C values ranged from -272‰ to -185‰ equivalent to calibrated ages of 1,100-2,550 years old. These ages may be the result of fractional preservation of biogenic carbon and/or sudden large-scale community death and extended period(s) of inactivity prior to slow recolonization and incorporation of 14 C-depleted fossil material. PLFA Δ14 C values were generally more modern than the corresponding TOC and varied widely between sites; the FV PLFA Δ14 C value (+40‰) was consistent with modern atmospheric CO2 , while UV values ranged from -199‰ to -79‰ (calibrated ages of 1,665-610 years). The observed variability in PLFA Δ14 C depletions is hypothesized to reflect variations in the extent of fixation of modern atmospheric CO2 and the preservation and recycling of older organic carbon by the community in various stages of sandstone recolonization. PLFA profiles and microbial community compositions as determined by molecular genetic characterizations and microscopy differed between the two valleys (e.g., predominance of biomarker 18:2 [>50%] in FV compared to UV), representing microbial communities that may reflect distinct stages of sandstone recolonization and/or environmental conditions. It is thus proposed that Dry Valley cryptoendolithic microbial communities are faster growing than previously estimated.

In Situ Field Sequencing and Life Detection in Remote (79°26'N) Canadian High Arctic Permafrost Ice Wedge Microbial Communities.

Significant progress is being made in the development of the next generation of low cost life detection instrumentation with much smaller size, mass and energy requirements. Here, we describe in situ life detection and sequencing in the field in soils over laying ice wedges in polygonal permafrost terrain on Axel Heiberg Island, located in the Canadian high Arctic (79°26'N), an analog to the polygonal permafrost terrain observed on Mars. The life detection methods used here include (1) the cryo-iPlate for culturing microorganisms using diffusion of in situ nutrients into semi-solid media (2) a Microbial Activity Microassay (MAM) plate (BIOLOG Ecoplate) for detecting viable extant microorganisms through a colourimetric assay, and (3) the Oxford Nanopore MinION for nucleic acid detection and sequencing of environmental samples and the products of MAM plate and cryo-iPlate. We obtained 39 microbial isolates using the cryo-iPlate, which included several putatively novel strains based on the 16S rRNA gene, including a Pedobacter sp. (96% closest similarity in GenBank) which we partially genome sequenced using the MinION. The MAM plate successfully identified an active community capable of L-serine metabolism, which was used for metagenomic sequencing with the MinION to identify the active and enriched community. A metagenome on environmental ice wedge soil samples was completed, with base calling and uplink/downlink carried out via satellite internet. Validation of MinION sequencing using the Illumina MiSeq platform was consistent with the results obtained with the MinION. The instrumentation and technology utilized here is pre-existing, low cost, low mass, low volume, and offers the prospect of equipping micro-rovers and micro-penetrators with aggressive astrobiological capabilities. Since potentially habitable astrobiology targets have been identified (RSLs on Mars, near subsurface water ice on Mars, the plumes and oceans of Europa and Enceladus), future astrobiology missions will certainly target these areas and there is a need for direct life detection instrumentation.

PacBio SMRT assembly of a complex multi-replicon genome reveals chlorocatechol degradative operon in a region of genome plasticity.

We have sequenced a Burkholderia genome that contains multiple replicons and large repetitive elements that would make it inherently difficult to assemble by short read sequencing technologies. We illustrate how the integrated long read correction algorithms implemented through the PacBio Single Molecule Real-Time (SMRT) sequencing technology successfully provided a de novo assembly that is a reasonable estimate of both the gene content and genome organization without making any further modifications. This assembly is comparable to related organisms assembled by more labour intensive methods. Our assembled genome revealed regions of genome plasticity for further investigation, one of which harbours a chlorocatechol degradative operon highly homologous to those previously identified on globally ubiquitous plasmids. In an ideal world, this assembly would still require experimental validation to confirm gene order and copy number of repeated elements. However, we submit that particularly in instances where a polished genome is not the primary goal of the sequencing project, PacBio SMRT sequencing provides a financially viable option for generating a biologically relevant genome estimate that can be utilized by other researchers for comparative studies.