Catchment characteristics and seasonality control the composition of microbial assemblages exported from three outlet glaciers of the Greenland Ice Sheet.

Glacial meltwater drains into proglacial rivers where it interacts with the surrounding landscape, collecting microbial cells as it travels downstream. Characterizing the composition of the resulting microbial assemblages in transport can inform us about intra-annual changes in meltwater flowpaths beneath the glacier as well as hydrological connectivity with proglacial areas. Here, we investigated how the structure of suspended microbial assemblages evolves over the course of a melt season for three proglacial catchments of the Greenland Ice Sheet (GrIS), reasoning that differences in glacier size and the proportion of glacierized versus non-glacierized catchment areas will influence both the identity and relative abundance of microbial taxa in transport. Streamwater samples were taken at the same time each day over a period of 3 weeks (summer 2018) to identify temporal patterns in microbial assemblages for three outlet glaciers of the GrIS, which differed in glacier size (smallest to largest; Russell, Leverett, and Isunnguata Sermia [IS]) and their glacierized: proglacial catchment area ratio (Leverett, 76; Isunnguata Sermia, 25; Russell, 2). DNA was extracted from samples, and 16S rRNA gene amplicons sequenced to characterize the structure of assemblages. We found that microbial diversity was significantly greater in Isunnguata Sermia and Russell Glacier rivers compared to Leverett Glacier, the latter of which having the smallest relative proglacial catchment area. Furthermore, the microbial diversity of the former two catchments continued to increase over monitored period, presumably due to increasing hydrologic connectivity with proglacial habitats. Meanwhile, diversity decreased over the monitored period in Leverett, which may have resulted from the evolution of an efficient subglacial drainage system. Linear discriminant analysis further revealed that bacteria characteristic to soils were disproportionately represented in the Isunnguata Sermia river, while putative methylotrophs were disproportionately abundant in Russell Glacier. Meanwhile, taxa typical for glacierized habitats (i.e., Rhodoferax and Polaromonas) dominated in the Leverett Glacier river. Our findings suggest that the proportion of deglaciated catchment area is more influential to suspended microbial assemblage structure than absolute glacier size, and improve our understanding of hydrological flowpaths, particulate entrainment, and transport.

Microbial genomics amidst the Arctic crisis.

The Arctic is warming - fast. Microbes in the Arctic play pivotal roles in feedbacks that magnify the impacts of Arctic change. Understanding the genome evolution, diversity and dynamics of Arctic microbes can provide insights relevant for both fundamental microbiology and interdisciplinary Arctic science. Within this synthesis, we highlight four key areas where genomic insights to the microbial dimensions of Arctic change are urgently required: the changing Arctic Ocean, greenhouse gas release from the thawing permafrost, 'biological darkening' of glacial surfaces, and human activities within the Arctic. Furthermore, we identify four principal challenges that provide opportunities for timely innovation in Arctic microbial genomics. These range from insufficient genomic data to develop unifying concepts or model organisms for Arctic microbiology to challenges in gaining authentic insights to the structure and function of low-biomass microbiota and integration of data on the causes and consequences of microbial feedbacks across scales. We contend that our insights to date on the genomics of Arctic microbes are limited in these key areas, and we identify priorities and new ways of working to help ensure microbial genomics is in the vanguard of the scientific response to the Arctic crisis.

Glacial microbiota are hydrologically connected and temporally variable.

Glaciers are melting rapidly. The concurrent export of microbial assemblages alongside glacial meltwater is expected to impact the ecology of adjoining ecosystems. Currently, the source of exported assemblages is poorly understood, yet this information may be critical for understanding how current and future glacial melt seasons may influence downstream environments. We report on the connectivity and temporal variability of microbiota sampled from supraglacial, subglacial and periglacial habitats and water bodies within a glacial catchment. Sampled assemblages showed evidence of being biologically connected through hydrological flowpaths, leading to a meltwater system that accumulates prokaryotic biota as it travels downstream. Temporal changes in the connected assemblages were similarly observed. Snow assemblages changed markedly throughout the sample period, likely reflecting changes in the surrounding environment. Changes in supraglacial meltwater assemblages reflected the transition of the glacial surface from snow-covered to bare-ice. Marked snowmelt across the surrounding periglacial environment resulted in the flushing of soil assemblages into the riverine system. In contrast, surface ice within the ablation zone and subglacial meltwaters remained relatively stable throughout the sample period. Our results are indicative that changes in snow and ice melt across glacial environments will influence the abundance and diversity of microbial assemblages transported downstream.

Patterns in Microbial Assemblages Exported From the Meltwater of Arctic and Sub-Arctic Glaciers.

Meltwater streams connect the glacial cryosphere with downstream ecosystems. Dissolved and particulate matter exported from glacial ecosystems originates from contrasting supraglacial and subglacial environments, and exported microbial cells have the potential to serve as ecological and hydrological indicators for glacial ecosystem processes. Here, we compare exported microbial assemblages from the meltwater of 24 glaciers from six (sub)Arctic regions - the southwestern Greenland Ice Sheet, Qeqertarsuaq (Disko Island) in west Greenland, Iceland, Svalbard, western Norway, and southeast Alaska - differing in their lithology, catchment size, and climatic characteristics, to investigate spatial and environmental factors structuring exported meltwater assemblages. We found that 16S rRNA gene sequences of all samples were dominated by the phyla Proteobacteria, Bacteroidetes, and Actinobacteria, with Verrucomicrobia also common in Greenland localities. Clustered OTUs were largely composed of aerobic and anaerobic heterotrophs capable of degrading a wide variety of carbon substrates. A small number of OTUs dominated all assemblages, with the most abundant being from the genera Polaromonas, Methylophilus, and Nitrotoga. However, 16-32% of a region's OTUs were unique to that region, and rare taxa revealed unique metabolic potentials and reflected differences between regions, such as the elevated relative abundances of sulfur oxidizers Sulfuricurvum sp. and Thiobacillus sp. at Svalbard sites. Meltwater alpha diversity showed a pronounced decrease with increasing latitude, and multivariate analyses of assemblages revealed significant regional clusters. Distance-based redundancy and correlation analyses further resolved associations between whole assemblages and individual OTUs with variables primarily corresponding with the sampled regions. Interestingly, some OTUs indicating specific metabolic processes were not strongly associated with corresponding meltwater characteristics (e.g., nitrification and inorganic nitrogen concentrations). Thus, while exported assemblage structure appears regionally specific, and probably reflects differences in dominant hydrological flowpaths, OTUs can also serve as indicators for more localized microbially mediated processes not captured by the traditional characterization of bulk meltwater hydrochemistry. These results collectively promote a better understanding of microbial distributions across the Arctic, as well as linkages between the terrestrial cryosphere habitats and downstream ecosystems.

Illuminating the dynamic rare biosphere of the Greenland Ice Sheet's Dark Zone.

Greenland's Dark Zone is the largest contiguous region of bare terrestrial ice in the Northern Hemisphere and microbial processes play an important role in driving its darkening and thereby amplifying melt and runoff from the ice sheet. However, the dynamics of these microbiota have not been fully identified. Here, we present joint 16S rRNA gene and 16S rRNA (cDNA) comparison of input (snow), storage (cryoconite) and output (supraglacial stream water) habitats across the Dark Zone over the melt season. We reveal that all three Dark Zone communities have a preponderance of rare taxa exhibiting high protein synthesis potential (PSP). Furthermore, taxa with high PSP represent highly connected 'bottlenecks' within community structure, consistent with their roles as metabolic hubs. Finally, low abundance-high PSP taxa affiliated with Methylobacterium within snow and stream water suggest a novel role for Methylobacterium in the carbon cycle of Greenlandic snowpacks, and importantly, the export of potentially active methylotrophs to the bed of the Greenland Ice Sheet. By comparing the dynamics of bulk and potentially active microbiota in the Dark Zone of the Greenland Ice Sheet, we provide novel insights into the mechanisms and impacts of the microbial colonization of this critical region of our melting planet.

Glacier Algae: A Dark Past and a Darker Future.

"Glacier algae" grow on melting glacier and ice sheet surfaces across the cryosphere, causing the ice to absorb more solar energy and consequently melt faster, while also turning over carbon and nutrients. This makes glacier algal assemblages, which are typically dominated by just three main species, a potentially important yet under-researched component of the global biosphere, carbon, and water cycles. This review synthesizes current knowledge on glacier algae phylogenetics, physiology, and ecology. We discuss their significance for the evolution of early land plants and highlight their impacts on the physical and chemical supraglacial environment including their role as drivers of positive feedbacks to climate warming, thereby demonstrating their influence on Earth's past and future. Four complementary research priorities are identified, which will facilitate broad advances in glacier algae research, including establishment of reliable culture collections, sequencing of glacier algae genomes, development of diagnostic biosignatures for remote sensing, and improved predictive modeling of glacier algae biological-albedo effects.

Potential Activity of Subglacial Microbiota Transported to Anoxic River Delta Sediments.

The Watson River drains a portion of the SW Greenland ice sheet, transporting microbial communities from subglacial environments to a delta at the head of Søndre Strømfjord. This study investigates the potential activity and community shifts of glacial microbiota deposited and buried under layers of sediments within the river delta. A long-term (12-month) incubation experiment was established using Watson River delta sediment under anaerobic conditions, with and without CO2/H2 enrichment. Within CO2/H2-amended incubations, sulphate depletion and a shift in the microbial community to a 52% predominance of Desulfosporosinus meridiei by day 371 provides evidence for sulphate reduction. We found evidence of methanogenesis in CO2/H2-amended incubations within the first 5 months, with production rates of ~4 pmol g-1 d-1, which was likely performed by methanogenic Methanomicrobiales- and Methanosarcinales-related organisms. Later, a reduction in methane was observed to be paired with the depletion of sulphate, and we hypothesise that sulphate reduction out competed hydrogenotrophic methanogenesis. The structure and diversity of the original CO2/H2-amended incubation communities changed dramatically with a major shift in predominant community members and a decline in diversity and cell abundance. These results highlight the need for further investigations into the fate of subglacial microbiota within downstream environments.

Nitrate addition has minimal short-term impacts on greenland ice sheet supraglacial prokaryotes.

Tropospheric nitrate levels are predicted to increase throughout the 21st century, with potential effects on terrestrial ecosystems, including the Greenland ice sheet (GrIS). This study considers the impacts of elevated nitrate concentrations on the abundance and composition of dominant bulk and active prokaryotic communities sampled from in situ nitrate fertilization plots on the GrIS surface. Nitrate concentrations were successfully elevated within sediment-filled meltwater pools, known as cryoconite holes; however, nitrate additions applied to surface ice did not persist. Estimated bulk and active cryoconite community cell abundance was unaltered by nitrate additions when compared to control holes using a quantitative PCR approach, and nitrate was found to have a minimal affect on the dominant 16S rRNA gene-based community composition. Together, these results indicate that sampled cryoconite communities were not nitrate limited at the time of sampling. Instead, temporal changes in biomass and community composition were more pronounced. As these in situ incubations were short (6 weeks), and the community composition across GrIS surface ice is highly variable, we suggest that further efforts should be considered to investigate the potential long-term impacts of increased nitrate across the GrIS.

Meltwater export of prokaryotic cells from the Greenland ice sheet.

Microorganisms are flushed from the Greenland Ice Sheet (GrIS) where they may contribute towards the nutrient cycling and community compositions of downstream ecosystems. We investigate meltwater microbial assemblages as they exit the GrIS from a large outlet glacier, and as they enter a downstream river delta during the record melt year of 2012. Prokaryotic abundance, flux and community composition was studied, and factors affecting community structures were statistically considered. The mean concentration of cells exiting the ice sheet was 8.30 × 104 cells mL-1 and we estimate that ∼1.02 × 1021 cells were transported to the downstream fjord in 2012, equivalent to 30.95 Mg of carbon. Prokaryotic microbial assemblages were dominated by Proteobacteria, Bacteroidetes, and Actinobacteria. Cell concentrations and community compositions were stable throughout the sample period, and were statistically similar at both sample sites. Based on our observations, we argue that the subglacial environment is the primary source of the river-transported microbiota, and that cell export from the GrIS is dependent on discharge. We hypothesise that the release of subglacial microbiota to downstream ecosystems will increase as freshwater flux from the GrIS rises in a warming world.

Metabolome-mediated biocryomorphic evolution promotes carbon fixation in Greenlandic cryoconite holes.

Microbial photoautotrophs on glaciers engineer the formation of granular microbial-mineral aggregates termed cryoconite which accelerate ice melt, creating quasi-cylindrical pits called 'cryoconite holes'. These act as biogeochemical reactors on the ice surface and provide habitats for remarkably active and diverse microbiota. Evolution of cryoconite holes towards an equilibrium depth is well known, yet interactions between microbial activity and hole morphology are currently weakly addressed. Here, we experimentally perturbed the depths and diameters of cryoconite holes on the Greenland Ice Sheet. Cryoconite holes responded by sensitively adjusting their shapes in three dimensions ('biocryomorphic evolution') thus maintaining favourable conditions for net autotrophy at the hole floors. Non-targeted metabolomics reveals concomitant shifts in cyclic AMP and fucose metabolism consistent with phototaxis and extracellular polymer synthesis indicating metabolomic-level granular changes in response to perturbation. We present a conceptual model explaining this process and suggest that it results in remarkably robust net autotrophy on the Greenland Ice Sheet. We also describe observations of cryoconite migrating away from shade, implying a degree of self-regulation of carbon budgets over mesoscales. Since cryoconite is a microbe-mineral aggregate, it appears that microbial processes themselves form and maintain stable autotrophic habitats on the surface of the Greenland ice sheet.

Supraglacial bacterial community structures vary across the Greenland ice sheet.

The composition and spatial variability of microbial communities that reside within the extensive (>200 000 km(2)) biologically active area encompassing the Greenland ice sheet (GrIS) is hypothesized to be variable. We examined bacterial communities from cryoconite debris and surface ice across the GrIS, using sequence analysis and quantitative PCR of 16S rRNA genes from co-extracted DNA and RNA. Communities were found to differ across the ice sheet, with 82.8% of the total calculated variation attributed to spatial distribution on a scale of tens of kilometers separation. Amplicons related to Sphingobacteriaceae, Pseudanabaenaceae and WPS-2 accounted for the greatest portion of calculated dissimilarities. The bacterial communities of ice and cryoconite were moderately similar (global R = 0.360, P = 0.002) and the sampled surface type (ice versus cryoconite) did not contribute heavily towards community dissimilarities (2.3% of total variability calculated). The majority of dissimilarities found between cryoconite 16S rRNA gene amplicons from DNA and RNA was calculated to be the result of changes in three taxa, Pseudanabaenaceae, Sphingobacteriaceae and WPS-2, which together contributed towards 80.8 ± 12.6% of dissimilarities between samples. Bacterial communities across the GrIS are spatially variable active communities that are likely influenced by localized biological inputs and physicochemical conditions.

Microbial abundance in surface ice on the Greenland Ice Sheet.

Measuring microbial abundance in glacier ice and identifying its controls is essential for a better understanding and quantification of biogeochemical processes in glacial ecosystems. However, cell enumeration of glacier ice samples is challenging due to typically low cell numbers and the presence of interfering mineral particles. We quantified for the first time the abundance of microbial cells in surface ice from geographically distinct sites on the Greenland Ice Sheet (GrIS), using three enumeration methods: epifluorescence microscopy (EFM), flow cytometry (FCM), and quantitative polymerase chain reaction (qPCR). In addition, we reviewed published data on microbial abundance in glacier ice and tested the three methods on artificial ice samples of realistic cell (10(2)-10(7) cells ml(-1)) and mineral particle (0.1-100 mg ml(-1)) concentrations, simulating a range of glacial ice types, from clean subsurface ice to surface ice to sediment-laden basal ice. We then used multivariate statistical analysis to identify factors responsible for the variation in microbial abundance on the ice sheet. EFM gave the most accurate and reproducible results of the tested methodologies, and was therefore selected as the most suitable technique for cell enumeration of ice containing dust. Cell numbers in surface ice samples, determined by EFM, ranged from ~ 2 × 10(3) to ~ 2 × 10(6) cells ml(-1) while dust concentrations ranged from 0.01 to 2 mg ml(-1). The lowest abundances were found in ice sampled from the accumulation area of the ice sheet and in samples affected by fresh snow; these samples may be considered as a reference point of the cell abundance of precipitants that are deposited on the ice sheet surface. Dust content was the most significant variable to explain the variation in the abundance data, which suggests a direct association between deposited dust particles and cells and/or by their provision of limited nutrients to microbial communities on the GrIS.

Diversity and potential sources of microbiota associated with snow on western portions of the Greenland Ice Sheet.

Snow overlays the majority of the Greenland Ice Sheet (GrIS). However, there is very little information available on the microbiological assemblages that are associated with this vast and climate-sensitive landscape. In this study, the structure and diversity of snow microbial assemblages from two regions of the western GrIS ice margin were investigated through the sequencing of small subunit ribosomal RNA genes. The origins of the microbiota were investigated by examining correlations to molecular data obtained from marine, soil, freshwater and atmospheric environments and geochemical analytes measured in the snow. Snow was found to contain a diverse assemblage of bacteria (Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria) and eukarya (Alveolata, Fungi, Stramenopiles and Chloroplastida). Phylotypes related to archaeal Thaumarchaeota and Euryarchaeota phyla were also identified. The snow microbial assemblages were more similar to communities characterized in soil than to those documented in marine ecosystems. Despite this, the chemical composition of snow samples was consistent with a marine contribution, and strong correlations existed between bacterial beta diversity and the concentration of Na(+) and Cl(-) . These results suggest that surface snow from western regions of Greenland contains exogenous microbiota that were likely aerosolized from more distant soil sources, transported in the atmosphere and co-precipitated with the snow.

Proteomics of Colwellia psychrerythraea at subzero temperatures - a life with limited movement, flexible membranes and vital DNA repair.

The mechanisms that allow psychrophilic bacteria to remain metabolically active at subzero temperatures result from form and function of their proteins. We present first proteomic evidence of physiological changes of the marine psychrophile Colwellia psychrerythraea 34H (Cp34H) after exposure to subzero temperatures (-1, and -10°C in ice) through 8 weeks. Protein abundance was compared between different treatments to understand the effects of temperature and time, independently and jointly, within cells transitioning to, and being maintained in ice. Parallel [3H]-leucine and [3H]-thymidine incubations indicated active protein and DNA synthesis to -10°C. Mass spectrometry-based proteomics identified 1763 proteins across four experimental treatments. Proteins involved in osmolyte regulation and polymer secretion were found constitutively present across all treatments, suggesting that they are required for metabolic success below 0°C. Differentially abundant protein groups indicated a reallocation of resources from DNA binding to DNA repair and from motility to chemo-taxis and sensing. Changes to iron and nitrogen metabolism, cellular membrane structures, and protein synthesis and folding were also revealed. By elucidating vital strategies during life in ice, this study provides novel insight into the extensive molecular adaptations that occur in cold-adapted marine organisms to sustain cellular function in their habitat.

Different bulk and active bacterial communities in cryoconite from the margin and interior of the Greenland ice sheet.

Biological processes in the supraglacial ecosystem, including cryoconite, contribute to nutrient cycling within the cryosphere and may affect surface melting, yet little is known of the diversity of the active microbes in these environments. We examined the bacterial abundance and community composition of cryoconite over a melt season at two contrasting sites at the margin and in the interior of the Greenland ice sheet, using sequence analysis and quantitative polymerase chain reaction of coextracted 16S rDNA and rRNA. Significant differences were found between bulk (rDNA) and potentially active (rRNA) communities, and between communities sampled from the two sites. Higher concentrations of rRNA than rDNA were detected at the interior site, whereas at the margin several orders of magnitude less rRNA was found compared with rDNA, which may be explained by a lower proportion of active bacteria at the margin site. The rRNA communities at both sites were dominated by a few taxa of Cyanobacteria and Alpha- and/or Betaproteobacteria. The bulk alpha diversity was higher in the margin site community, suggesting that local sources may be contributing towards the gene pool in addition to long distance transport.

Molecular and biogeochemical evidence for methane cycling beneath the western margin of the Greenland Ice Sheet.

Microbial processes that mineralize organic carbon and enhance solute production at the bed of polar ice sheets could be of a magnitude sufficient to affect global elemental cycles. To investigate the biogeochemistry of a polar subglacial microbial ecosystem, we analyzed water discharged during the summer of 2012 and 2013 from Russell Glacier, a land-terminating outlet glacier at the western margin of the Greenland Ice Sheet. The molecular data implied that the most abundant and active component of the subglacial microbial community at these marginal locations were bacteria within the order Methylococcales (59-100% of reverse transcribed (RT)-rRNA sequences). mRNA transcripts of the particulate methane monooxygenase (pmoA) from these taxa were also detected, confirming that methanotrophic bacteria were functional members of this subglacial ecosystem. Dissolved methane ranged between 2.7 and 83 µM in the subglacial waters analyzed, and the concentration was inversely correlated with dissolved oxygen while positively correlated with electrical conductivity. Subglacial microbial methane production was supported by δ(13)C-CH4 values between -64‰ and -62‰ together with the recovery of RT-rRNA sequences that classified within the Methanosarcinales and Methanomicrobiales. Under aerobic conditions, >98% of the methane in the subglacial water was consumed over ∼30 days incubation at ∼4 °C and rates of methane oxidation were estimated at 0.32 µM per day. Our results support the occurrence of active methane cycling beneath this region of the Greenland Ice Sheet, where microbial communities poised in oxygenated subglacial drainage channels could serve as significant methane sinks.

Structure and diversity of bacterial, eukaryotic and archaeal communities in glacial cryoconite holes from the Arctic and the Antarctic.

The cryosphere presents some of the most challenging conditions for life on earth. Nevertheless, (micro)biota survive in a range of niches in glacial systems, including water-filled depressions on glacial surfaces termed cryoconite holes (centimetre to metre in diameter and up to 0.5 m deep) that contain dark granular material (cryoconite). In this study, the structure of bacterial and eukaryotic cryoconite communities from ten different locations in the Arctic and Antarctica was compared using T-RFLP analysis of rRNA genes. Community structure varied with geography, with greatest differences seen between communities from the Arctic and the Antarctic. DNA sequencing of rRNA genes revealed considerable diversity, with individual cryoconite hole communities containing between six and eight bacterial phyla and five and eight eukaryotic 'first-rank' taxa and including both bacterial and eukaryotic photoautotrophs. Bacterial Firmicutes and Deltaproteobacteria and Epsilonproteobacteria, eukaryotic Rhizaria, Haptophyta, Choanomonada and Centroheliozoa, and archaea were identified for the first time in cryoconite ecosystems. Archaea were only found within Antarctic locations, with the majority of sequences (77%) related to members of the Thaumarchaeota. In conclusion, this research has revealed that Antarctic and Arctic cryoconite holes harbour geographically distinct highly diverse communities and has identified hitherto unknown bacterial, eukaryotic and archaeal taxa, therein.