Characterization of hydrocarbon degraders from Northwest Passage beach sediments and assessment of their ability for bioremediation.

Global warming-induced sea ice loss in the Canadian Northwest Passage (NWP) will result in more shipping traffic, increasing the risk of oil spills. Microorganisms inhabiting NWP beach sediments may degrade hydrocarbons, offering a potential bioremediation strategy. In this study, the characterization and genomic analyses of 22 hydrocarbon-biodegradative bacterial isolates revealed that they contained a diverse range of key alkane and aromatic hydrocarbon-degradative genes, as well as cold and salt tolerance genes indicating they are highly adapted to the extreme Arctic environment. Some isolates successfully degraded Ultra Low Sulfur Fuel Oil (ULSFO) at temperatures as low as -5 °C and high salinities (3%-10%). Three isolates were grown in liquid medium containing ULSFO as sole carbon source over 3 months and variation of hydrocarbon concentration was measured at three time points to determine their rate of hydrocarbon biodegradation. Our results demonstrate that two isolates (Rhodococcus sp. R1B_2T and Pseudarthrobacter sp. R2D_1T) possess complete degradation pathways and can grow on alkane and aromatic components of ULSFO under Arctic conditions. Overall, these results demonstrate that diverse hydrocarbon-degrading microorganisms exist in the NWP beach sediments, offering a potential bioremediation strategy in the events of a marine fuel spill reaching the shores of the NWP.

Thiomicrorhabdus streamers and sulfur cycling in perennial hypersaline cold springs in the Canadian high Arctic.

The Gypsum Hill (GH) springs on Axel Heiberg Island in the Canadian high Arctic are host to chemolithoautotrophic, sulfur-oxidizing streamers that flourish in the high Arctic winter in water temperatures from -1.3 to 7°C with ~8% salinity in a high Arctic winter environment with air temperatures commonly less than -40°C and an average annual air temperature of -15°C. Metagenome sequencing and binning of streamer samples produced a 96% complete Thiomicrorhabdus sp. metagenome-assembled genome representing a possible new species or subspecies. This is the most cold- and salt-extreme source environment for a Thiomicrorhabdus genome yet described. Metaproteomic and metatranscriptomic analysis attributed nearly all gene expression in the streamers to the Thiomicrorhabdus sp. and suggested that it is active in CO2 fixation and oxidation of sulfide to elemental sulfur. In situ geochemical and isotopic analyses of the fractionation of multiple sulfur isotopes determined the biogeochemical transformation of sulfur from its source in Carboniferous evaporites to biotic processes occurring in the sediment and streamers. These complementary molecular tools provided a functional link between the geochemical substrates and the collective traits and activity that define the microbial community's interactions within a unique polar saline habitat where Thiomicrorhabdus-dominated streamers form and flourish.

Species interactions and distinct microbial communities in high Arctic permafrost affected cryosols are associated with the CH4 and CO2 gas fluxes.

Microbial metabolism of the thawing organic carbon stores in permafrost results in a positive feedback loop of greenhouse gas emissions. CO2 and CH4 fluxes and the associated microbial communities in Arctic cryosols are important in predicting future warming potential of the Arctic. We demonstrate that topography had an impact on CH4 and CO2 flux at a high Arctic ice-wedge polygon terrain site, with higher CO2 emissions and lower CH4 uptake at troughs compared to polygon interior soils. The pmoA sequencing suggested that USCα cluster of uncultured methanotrophs is likely responsible for observed methane sink. Community profiling revealed distinct assemblages across the terrain at different depths. Deeper soils contained higher abundances of Verrucomicrobia and Gemmatimonadetes, whereas the polygon interior had higher Acidobacteria and lower Betaproteobacteria and Deltaproteobacteria abundances. Genome sequencing of isolates from the terrain revealed presence of carbon cycling genes including ones involved in serine and ribulose monophosphate pathways. A novel hybrid network analysis identified key members that had positive and negative impacts on other species. Operational Taxonomic Units (OTUs) with numerous positive interactions corresponded to Proteobacteria, Candidatus Rokubacteria and Actinobacteria phyla, while Verrucomicrobia and Acidobacteria members had negative impacts on other species. Results indicate that topography and microbial interactions impact community composition.

Comparative activity and functional ecology of permafrost soils and lithic niches in a hyper-arid polar desert.

Permafrost in the high elevation McMurdo Dry Valleys of Antarctica ranks among the driest and coldest on Earth. Permafrost soils appear to be largely inhospitable to active microbial life, but sandstone lithic microhabitats contain a trophically simple but functional cryptoendolithic community. We used metagenomic sequencing and activity assays to examine the functional capacity of permafrost soils and cryptoendolithic communities in University Valley, one of the most extreme regions in the Dry Valleys. We found metagenomic evidence that cryptoendolithic microorganisms are adapted to the harsh environment and capable of metabolic activity at in situ temperatures, possessing a suite of stress response and nutrient cycling genes to fix carbon under the fluctuating conditions that the sandstone rock would experience during the summer months. We additionally identified genes involved in microbial competition and cooperation within the cryptoendolithic habitat. In contrast, permafrost soils have a lower richness of stress response genes, and instead the metagenome is enriched in genes involved with dormancy and sporulation. The permafrost soils also have a large presence of phage genes and genes involved in the recycling of cellular material. Our results underlie two different habitability conditions under extreme cold and dryness: the permafrost soil which is enriched in traits which emphasize survival and dormancy, rather than growth and activity; and the cryptoendolithic environment that selects for organisms capable of growth under extremely oligotrophic, arid and cold conditions. This study represents the first metagenomic interrogation of Antarctic permafrost and polar cryptoendolithic microbial communities.

Mechanisms of subzero growth in the cryophile Planococcus halocryophilus determined through proteomic analysis.

The eurypsychrophilic bacterium Planococcus halocryophilus is capable of growth down to -15°C, making it ideal for studying adaptations to subzero growth. To increase our understanding of the mechanisms and pathways important for subzero growth, we performed proteomics on P. halocryophilus grown at 23°C, 23°C with 12% w/v NaCl and -10°C with 12% w/v NaCl. Many proteins with increased abundances at -10°C versus 23°C also increased at 23C-salt versus 23°C, indicating a closely tied relationship between salt and cold stress adaptation. Processes which displayed the largest changes in protein abundance were peptidoglycan and fatty acid (FA) synthesis, translation processes, methylglyoxal metabolism, DNA repair and recombination, and protein and nucleotide turnover. We identified intriguing targets for further research at -10°C, including PlsX and KASII (FA metabolism), DD-transpeptidase and MurB (peptidoglycan synthesis), glyoxalase family proteins (reactive electrophile response) and ribosome modifying enzymes (translation turnover). PemK/MazF may have a crucial role in translational reprogramming under cold conditions. At -10°C P. halocryophilus induces stress responses, uses resources efficiently, and carefully controls its growth and metabolism to maximize subzero survival. The present study identifies several mechanisms involved in subzero growth and enhances our understanding of cold adaptation.

Sulfur isotope fractionation during the evolutionary adaptation of a sulfate-reducing bacterium.

Dissimilatory sulfate reduction is a microbial catabolic pathway that preferentially processes less massive sulfur isotopes relative to their heavier counterparts. This sulfur isotope fractionation is recorded in ancient sedimentary rocks and generally is considered to reflect a phenotypic response to environmental variations rather than to evolutionary adaptation. Modern sulfate-reducing microorganisms isolated from similar environments can exhibit a wide range of sulfur isotope fractionations, suggesting that adaptive processes influence the sulfur isotope phenotype. To date, the relationship between evolutionary adaptation and isotopic phenotypes has not been explored. We addressed this by studying the covariation of fitness, sulfur isotope fractionation, and growth characteristics in Desulfovibrio vulgaris Hildenborough in a microbial evolution experiment. After 560 generations, the mean fitness of the evolved lineages relative to the starting isogenic population had increased by ∼ 17%. After 927 generations, the mean fitness relative to the initial ancestral population had increased by ∼ 20%. Growth rate in exponential phase increased during the course of the experiment, suggesting that this was a primary influence behind the fitness increases. Consistent changes were observed within different selection intervals between fractionation and fitness. Fitness changes were associated with changes in exponential growth rate but changes in fractionation were not. Instead, they appeared to be a response to changes in the parameters that govern growth rate: yield and cell-specific sulfate respiration rate. We hypothesize that cell-specific sulfate respiration rate, in particular, provides a bridge that allows physiological controls on fractionation to cross over to the adaptive realm.

Evidence of in situ microbial activity and sulphidogenesis in perennially sub-0 °C and hypersaline sediments of a high Arctic permafrost spring.

The lost hammer (LH) spring perennially discharges subzero hypersaline reducing brines through thick layers of permafrost and is the only known terrestrial methane seep in frozen settings on Earth. The present study aimed to identify active microbial communities that populate the sediments of the spring outlet, and verify whether such communities vary seasonally and spatially. Microcosm experiments revealed that the biological reduction of sulfur compounds (SR) with hydrogen (e.g., sulfate reduction) was potentially carried out under combined hypersaline and subzero conditions, down to -20 °C, the coldest temperature ever recorded for SR. Pyrosequencing analyses of both 16S rRNA (i.e., cDNA) and 16S rRNA genes (i.e., DNA) of sediments retrieved in late winter and summer indicated fairly stable bacterial and archaeal communities at the phylum level. Potentially active bacterial and archaeal communities were dominated by clades related to the T78 Chloroflexi group and Halobacteria species, respectively. The present study indicated that SR, hydrogenotrophy (possibly coupled to autotrophy), and short-chain alkane degradation (other than methane), most likely represent important, previously unaccounted for, metabolic processes carried out by LH microbial communities. Overall, the obtained findings provided additional evidence that the LH system hosts active communities of anaerobic, halophilic, and cryophilic microorganisms despite the extreme conditions in situ.

Characterizing the surface-exposed proteome of Planococcus halocryophilus during cryophilic growth.

Planococcus halocryophilus OR1 is a bacterial isolate capable of growth at temperatures ranging from -15 to +37 °C. During sub-zero (cryophilic) growth, nodular features appear on its cell surface; however, the biochemical compositions of these features as well as any cold-adaptive benefits they may offer are not understood. This study aimed to identify differences in the cell surface proteome (surfaceome) of P. halocryophilus cells grown under optimal (24 °C, no added salt), low- and mid-salt (5 and 12 % NaCl, respectively) at 24 °C, and low- and mid-salt sub-zero (5 % NaCl at -5 °C and 12 % NaCl at -10 °C) culture conditions, for the purpose of gaining insight into cold-adapted proteomic traits at the cell surface. Mid-log cells were harvested, treated briefly with trypsin and the resultant peptides were purified followed by identification by LC-MS/MS analysis. One hundred and forty-four proteins were subsequently identified in at least one culture condition. Statistically significant differences in amino acid usage, a known indicator of cold adaptation, were identified through in silico analysis. Two proteins with roles in peptidoglycan (PG) metabolism, an N-acetyl-L-alanine amidase and a multimodular transpeptidase-transglycosylase, were detected, though each was only detected under optimal conditions, indicating that high-salt and high-cold stress each affect PG metabolism. Two iron transport-binding proteins, associated with two different iron transport strategies, were identified, indicating that P. halocryophilus uses a different iron acquisition strategy at very low temperatures. Here we present the first set of data that describes bacterial adaptations at the cellular surface that occur as a cryophilic bacterium is transitioned from optimal to near-inhibitory sub-zero culture conditions.

High Arctic seawater and coastal soil microbiome co-occurrence and composition structure and their potential hydrocarbon biodegradation.

The accelerated decline in Arctic sea-ice cover and duration is enabling the opening of Arctic marine passages and improving access to natural resources. The increasing accessibility to navigation and resource exploration and production brings risks of accidental hydrocarbon releases into Arctic waters, posing a major threat to Arctic marine ecosystems where oil may persist for many years, especially in beach sediment. The composition and response of the microbial community to oil contamination on Arctic beaches remain poorly understood. To address this, we analyzed microbial community structure and identified hydrocarbon degradation genes among the Northwest Passage intertidal beach sediments and shoreline seawater from five high Arctic beaches. Our results from 16S/18S rRNA genes, long-read metagenomes, and metagenome-assembled genomes reveal the composition and metabolic capabilities of the hydrocarbon microbial degrader community, as well as tight cross-habitat and cross-kingdom interactions dominated by lineages that are common and often dominant in the polar coastal habitat, but distinct from petroleum hydrocarbon-contaminated sites. In the polar beach sediment habitats, Granulosicoccus sp. and Cyclocasticus sp. were major potential hydrocarbon-degraders, and our metagenomes revealed a small proportion of microalgae and algal viruses possessing key hydrocarbon biodegradative genes. This research demonstrates that Arctic beach sediment and marine microbial communities possess the ability for hydrocarbon natural attenuation. The findings provide new insights into the viral and microalgal communities possessing hydrocarbon degradation genes and might represent an important contribution to the removal of hydrocarbons under harsh environmental conditions in a pristine, cold, and oil-free environment that is threatened by oil spills.

Metagenomic survey reveals hydrocarbon biodegradation potential of Canadian high Arctic beaches.

BACKGROUND: Decreasing sea ice coverage across the Arctic Ocean due to climate change is expected to increase shipping activity through previously inaccessible shipping routes, including the Northwest Passage (NWP). Changing weather conditions typically encountered in the Arctic will still pose a risk for ships which could lead to an accident and the uncontrolled release of hydrocarbons onto NWP shorelines. We performed a metagenomic survey to characterize the microbial communities of various NWP shorelines and to determine whether there is a metabolic potential for hydrocarbon degradation in these microbiomes. RESULTS: We observed taxonomic and functional gene evidence supporting the potential of NWP beach microbes to degrade various types of hydrocarbons. The metagenomic and metagenome-assembled genome (MAG) taxonomy showed that known hydrocarbon-degrading taxa are present in these beaches. Additionally, we detected the presence of biomarker genes of aerobic and anaerobic degradation pathways of alkane and aromatic hydrocarbons along with complete degradation pathways for aerobic alkane degradation. Alkane degradation genes were present in all samples and were also more abundant (33.8 ± 34.5 hits per million genes, HPM) than their aromatic hydrocarbon counterparts (11.7 ± 12.3 HPM). Due to the ubiquity of MAGs from the genus Rhodococcus (23.8% of the MAGs), we compared our MAGs with Rhodococcus genomes from NWP isolates obtained using hydrocarbons as the carbon source to corroborate our results and to develop a pangenome of Arctic Rhodococcus. Our analysis revealed that the biodegradation of alkanes is part of the core pangenome of this genus. We also detected nitrogen and sulfur pathways as additional energy sources and electron donors as well as carbon pathways providing alternative carbon sources. These pathways occur in the absence of hydrocarbons allowing microbes to survive in these nutrient-poor beaches. CONCLUSIONS: Our metagenomic analyses detected the genetic potential for hydrocarbon biodegradation in these NWP shoreline microbiomes. Alkane metabolism was the most prevalent type of hydrocarbon degradation observed in these tidal beach ecosystems. Our results indicate that bioremediation could be used as a cleanup strategy, but the addition of adequate amounts of N and P fertilizers, should be considered to help bacteria overcome the oligotrophic nature of NWP shorelines.

Biosignature Detection and MinION Sequencing of Antarctic Cryptoendoliths After Exposure to Mars Simulation Conditions.

In the search for life in our Solar System, Mars remains a promising target based on its proximity and similarity to Earth. When Mars transitioned from a warmer, wetter climate to its current dry and freezing conditions, any putative extant life probably retreated into habitable refugia such as the subsurface or the interior of rocks. Terrestrial cryptoendolithic microorganisms (i.e., those inhabiting rock interiors) thus represent possible modern-day Mars analogs, particularly those from the hyperarid McMurdo Dry Valleys in Antarctica. As DNA is a strong definitive biosignature, given that there is no known abiotic chemistry that can polymerize nucleobases, we investigated DNA detection with MinION sequencing in Antarctic cryptoendoliths after an ∼58-sol exposure in MARTE, a Mars environmental chamber capable of simulating martian temperature, pressure, humidity, ultraviolet (UV) radiation, and atmospheric composition, in conjunction with protein and lipid detection. The MARTE conditions resulted in changes in community composition and DNA, proteins, and cell membrane-derived lipids remained detectable postexposure. Of the multitude of extreme environmental conditions on Mars, UV radiation (specifically UVC) is the most destructive to both cells and DNA. As such, we further investigated if a UVC exposure corresponding to ∼278 martian years would impede DNA detection via MinION sequencing. The MinION was able to successfully detect and sequence DNA after this UVC radiation exposure, suggesting its utility for life detection in future astrobiology missions focused on finding relatively recently exposed biomarkers inside possible martian refugia.

Defining the functional potential and active community members of a sediment microbial community in a high-arctic hypersaline subzero spring.

The Lost Hammer (LH) Spring is the coldest and saltiest terrestrial spring discovered to date and is characterized by perennial discharges at subzero temperatures (-5°C), hypersalinity (salinity, 24%), and reducing (≈-165 mV), microoxic, and oligotrophic conditions. It is rich in sulfates (10.0%, wt/wt), dissolved H2S/sulfides (up to 25 ppm), ammonia (≈381 µM), and methane (11.1 g day(-1)). To determine its total functional and genetic potential and to identify its active microbial components, we performed metagenomic analyses of the LH Spring outlet microbial community and pyrosequencing analyses of the cDNA of its 16S rRNA genes. Reads related to Cyanobacteria (19.7%), Bacteroidetes (13.3%), and Proteobacteria (6.6%) represented the dominant phyla identified among the classified sequences. Reconstruction of the enzyme pathways responsible for bacterial nitrification/denitrification/ammonification and sulfate reduction appeared nearly complete in the metagenomic data set. In the cDNA profile of the LH Spring active community, ammonia oxidizers (Thaumarchaeota), denitrifiers (Pseudomonas spp.), sulfate reducers (Desulfobulbus spp.), and other sulfur oxidizers (Thermoprotei) were present, highlighting their involvement in nitrogen and sulfur cycling. Stress response genes for adapting to cold, osmotic stress, and oxidative stress were also abundant in the metagenome. Comparison of the composition of the functional community of the LH Spring to metagenomes from other saline/subzero environments revealed a close association between the LH Spring and another Canadian high-Arctic permafrost environment, particularly in genes related to sulfur metabolism and dormancy. Overall, this study provides insights into the metabolic potential and the active microbial populations that exist in this hypersaline cryoenvironment and contributes to our understanding of microbial ecology in extreme environments.

Sulfur-cycling chemolithoautotrophic microbial community dominates a cold, anoxic, hypersaline Arctic spring.

BACKGROUND: Gypsum Hill Spring, located in Nunavut in the Canadian High Arctic, is a rare example of a cold saline spring arising through thick permafrost. It perennially discharges cold (~ 7 °C), hypersaline (7-8% salinity), anoxic (~ 0.04 ppm O2), and highly reducing (~ - 430 mV) brines rich in sulfate (2.2 g.L-1) and sulfide (9.5 ppm), making Gypsum Hill an analog to putative sulfate-rich briny habitats on extraterrestrial bodies such as Mars. RESULTS: Genome-resolved metagenomics and metatranscriptomics were utilized to describe an active microbial community containing novel metagenome-assembled genomes and dominated by sulfur-cycling Desulfobacterota and Gammaproteobacteria. Sulfate reduction was dominated by hydrogen-oxidizing chemolithoautotrophic Desulfovibrionaceae sp. and was identified in phyla not typically associated with sulfate reduction in novel lineages of Spirochaetota and Bacteroidota. Highly abundant and active sulfur-reducing Desulfuromusa sp. highly transcribed non-coding RNAs associated with transcriptional regulation, showing potential evidence of putative metabolic flexibility in response to substrate availability. Despite low oxygen availability, sulfide oxidation was primarily attributed to aerobic chemolithoautotrophic Halothiobacillaceae. Low abundance and transcription of photoautotrophs indicated sulfur-based chemolithoautotrophy drives primary productivity even during periods of constant illumination. CONCLUSIONS: We identified a rare surficial chemolithoautotrophic, sulfur-cycling microbial community active in a unique anoxic, cold, hypersaline Arctic spring. We detected Mars-relevant metabolisms including hydrogenotrophic sulfate reduction, sulfur reduction, and sulfide oxidation, which indicate the potential for microbial life in analogous S-rich brines on past and present Mars. Video Abstract.

Microbial Characterization of Arctic Glacial Ice Cores with a Semiautomated Life Detection System.

The search for extant microbial life will be a major focus of future astrobiology missions; however, no direct extant life detection instrumentation is included in current missions to Mars. In this study, we developed the semiautomated MicroLife detection platform that collects and processes environmental samples, detects biosignatures, and characterizes microbial activity. This platform is composed of a drill for sample collection, a redox dye colorimetric system for microbial metabolic activity detection and assessment (µMAMA [microfluidics Microbial Activity MicroAssay]), and a MinION sequencer for biosignature detection and characterization of microbial communities. The MicroLife platform was field-tested on White Glacier on Axel Heiberg Island in the Canadian high Arctic, with two extracted ice cores. The µMAMA successfully detected microbial metabolism from the ice cores within 1 day of incubation. The MinION sequencing of the ice cores and the positive µMAMA card identified a microbial community consistent with cold and oligotrophic environments. Furthermore, isolation and identification of microbial isolates from the µMAMA card corroborated the MinION sequencing. Together, these analyses support the MicroLife platform's efficacy in identifying microbes natively present in cryoenvironments and detecting their metabolic activity. Given our MicroLife platform's size and low energy requirements, it could be incorporated into a future landed platform or rovers for life detection.

Effects of marine diesel on microbial diversity and activity in high Arctic beach sediments.

Global warming induced sea ice loss increases Arctic maritime traffic, enhancing the risk of ecosystem contamination from fuel spills and nutrient loading. The impact of marine diesel on bacterial metabolic activity and diversity, assessed by colorimetric assay, 16S rRNA and metagenomic sequencing, of Northwest Passage (Arctic Ocean) beach sediments was assessed with nutrient amendment at environmentally relevant temperatures (5 and 15 °C). Higher temperature and nutrients stimulated microbial activity, while diesel reduced it, with metabolism inhibited at and above 0.01 % (without nutrients) and at 1 % (with nutrients) diesel inclusions. Diesel exposure significantly decreased microbial diversity and selected for Psychrobacter genus. Microbial hydrocarbon degradation, organic compound metabolism, and exopolysaccharide production gene abundances increased under higher diesel concentrations. Metagenomic binning recovered nine MAGs/bins with hydrocarbon degradation genes. We demonstrate a nutrients' rescue-type effect in diesel contaminated microbial communities via enrichment of microorganisms with stress response, aromatic compound, and ammonia assimilation metabolisms.

Planococcus halocryophilus sp. nov., an extreme sub-zero species from high Arctic permafrost.

A novel aerobic, Gram-positive, motile, coccoid bacterial strain, designated Or1(T), was isolated from permafrost active-layer soil collected from the Canadian high Arctic. Strain Or1(T) was capable of growth over a broad temperature range, including sub-zero growth (below -10 to 37 °C), and at high salinity (0-19% NaCl), growing optimally at 25 °C, at pH 7.0-8.0 and in the presence of 2% NaCl. Its taxonomic and phylogenetic position was determined by using a polyphasic approach, which indicated that strain Or1(T) was a member of the genus Planococcus. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain Or1(T) belonged to the genus Planococcus, differing by 0.4-3.6% from the type strains of all recognized Planococcus species, and was related most closely to Planococcus antarcticus CMS 26or(T) (98.8% similarity) and Planococcus donghaensis JH1(T) (99.6%). However, DNA-DNA hybridization experiments showed that strain Or1(T) had low genomic relatedness to Planococcus antarcticus CMS 26or(T) (18%) and Planococcus donghaensis JH1(T) (46%). The major menaquinones of strain Or1(T) were MK-7 (55%), MK-8 (36%) and MK-6 (9%) and the major fatty acids were anteiso-C(15:0), C(16:1)ω7c alcohol and anteiso-C(17:0). The DNA G+C content of strain Or1(T) was 40.5 mol%. Differential phenotypic, phylogenetic and genomic data suggest that strain Or1(T) represents a novel species of the genus Planococcus, for which the name Planococcus halocryophilus sp. nov. is proposed. The type strain is Or1(T) ( = DSM 24743(T) = JCM 17719(T)).

Microbial diversity and activity in hypersaline high Arctic spring channels.

Lost Hammer (LH) spring is a unique hypersaline, subzero, perennial high Arctic spring arising through thick permafrost. In the present study, the microbial and geochemical characteristics of the LH outflow channels, which remain unfrozen at ≥-18°C and are more aerobic/less reducing than the spring source were examined and compared to the previously characterized spring source environment. LH channel sediments contained greater microbial biomass (~100-fold) and greater microbial diversity reflected by the 16S rRNA clone libraries. Phylotypes related to methanogenesis, methanotrophy, sulfur reduction and oxidation were detected in the bacterial clone libraries while the archaeal community was dominated by phylotypes most closely related to THE ammonia-oxidizing Thaumarchaeota. The cumulative percent recovery of (14)C-acetate mineralization in channel sediment microcosms exceeded ~30% and ~10% at 5 and -5°C, respectively, but sharply decreased at -10°C (≤1%). Most bacterial isolates (Marinobacter, Planococcus, and Nesterenkonia spp.) were psychrotrophic, halotolerant, and capable of growth at -5°C. Overall, the hypersaline, subzero LH spring channel has higher microbial diversity and activity than the source, and supports a variety of niches reflecting the more dynamic and heterogeneous channel environment.

Unique high Arctic methane metabolizing community revealed through in situ 13CH4-DNA-SIP enrichment in concert with genome binning.

Greenhouse gas (GHG) emissions from Arctic permafrost soils create a positive feedback loop of climate warming and further GHG emissions. Active methane uptake in these soils can reduce the impact of GHG on future Arctic warming potential. Aerobic methane oxidizers are thought to be responsible for this apparent methane sink, though Arctic representatives of these organisms have resisted culturing efforts. Here, we first used in situ gas flux measurements and qPCR to identify relative methane sink hotspots at a high Arctic cytosol site, we then labeled the active microbiome in situ using DNA Stable Isotope Probing (SIP) with heavy 13CH4 (at 100 ppm and 1000 ppm). This was followed by amplicon and metagenome sequencing to identify active organisms involved in CH4 metabolism in these high Arctic cryosols. Sequencing of 13C-labeled pmoA genes demonstrated that type II methanotrophs (Methylocapsa) were overall the dominant active methane oxidizers in these mineral cryosols, while type I methanotrophs (Methylomarinovum) were only detected in the 100 ppm SIP treatment. From the SIP-13C-labeled DNA, we retrieved nine high to intermediate quality metagenome-assembled genomes (MAGs) belonging to the Proteobacteria, Gemmatimonadetes, and Chloroflexi, with three of these MAGs containing genes associated with methanotrophy. A novel Chloroflexi MAG contained a mmoX gene along with other methane oxidation pathway genes, identifying it as a potential uncultured methane oxidizer. This MAG also contained genes for copper import, synthesis of biopolymers, mercury detoxification, and ammonia uptake, indicating that this bacterium is strongly adapted to conditions in active layer permafrost and providing new insights into methane biogeochemical cycling. In addition, Betaproteobacterial MAGs were also identified as potential cross-feeders with methanotrophs in these Arctic cryosols. Overall, in situ SIP labeling combined with metagenomics and genome binning demonstrated to be a useful tool for discovering and characterizing novel organisms related to specific microbial functions or biogeochemical cycles of interest. Our findings reveal a unique and active Arctic cryosol microbial community potentially involved in CH4 cycling.

Active lithoautotrophic and methane-oxidizing microbial community in an anoxic, sub-zero, and hypersaline High Arctic spring.

Lost Hammer Spring, located in the High Arctic of Nunavut, Canada, is one of the coldest and saltiest terrestrial springs discovered to date. It perennially discharges anoxic (<1 ppm dissolved oxygen), sub-zero (~-5 °C), and hypersaline (~24% salinity) brines from the subsurface through up to 600 m of permafrost. The sediment is sulfate-rich (1 M) and continually emits gases composed primarily of methane (~50%), making Lost Hammer the coldest known terrestrial methane seep and an analog to extraterrestrial habits on Mars, Europa, and Enceladus. A multi-omics approach utilizing metagenome, metatranscriptome, and single-amplified genome sequencing revealed a rare surface terrestrial habitat supporting a predominantly lithoautotrophic active microbial community driven in part by sulfide-oxidizing Gammaproteobacteria scavenging trace oxygen. Genomes from active anaerobic methane-oxidizing archaea (ANME-1) showed evidence of putative metabolic flexibility and hypersaline and cold adaptations. Evidence of anaerobic heterotrophic and fermentative lifestyles were found in candidate phyla DPANN archaea and CG03 bacteria genomes. Our results demonstrate Mars-relevant metabolisms including sulfide oxidation, sulfate reduction, anaerobic oxidation of methane, and oxidation of trace gases (H2, CO2) detected under anoxic, hypersaline, and sub-zero ambient conditions, providing evidence that similar extant microbial life could potentially survive in similar habitats on Mars.

Assessment of Automated Nucleic Acid Extraction Systems in Combination with MinION Sequencing As Potential Tools for the Detection of Microbial Biosignatures.

The utilization of nanopore technologies for the detection of organic biogenic compounds has garnered significant focus in recent years. Oxford Nanopore Technologies' (ONT) MinION instrument, which can detect and sequence nucleic acids (NAs), is one such example. These technologies have much promise for unambiguous life detection but require significant development in terms of methods for extraction and preparation of NAs for biosignature detection and their feasibility for use in astrobiology-focused field missions. In this study, we tested pre-existing, automated, or semiautomated NA extraction technologies, coupled with automated ONT VolTRAX NA sample preparation, and verification with Nanopore MinION sequencing. All of the extraction systems tested (SuperFastPrep2, ClaremontX1, and SOLID-Sample Preparation Unit) showed potential for extracting DNA from Canadian High Arctic environments analogous to Mars, Europa, and Enceladus, which could subsequently be detected and sequenced with the MinION. However, they differed with regard to efficacy, yield, purity, and sequencing and annotation quality. Overall, bead beating-based systems performed the best for these parameters. In addition, we showed that the MinION could sequence unpurified DNA contained in crude cell lysates. This is valuable from an astrobiology perspective because purification steps are time-consuming and complicate the requirements for an automated extraction and life detection system. Our results indicate that semiautomated NA extraction and preparation technologies hold much promise, and with increased optimization and automation could be coupled to a larger platform incorporating nanopore detection and sequencing of NAs for life detection applications.