Fig 1.
Schematic of selected microbial habitats in the frozen environments of sea ice and permafrost.
(A) Sea ice (not drawn to scale). Microorganisms, in particular heterotrophic bacteria, inhabit all dimensions and seasons of sea ice and its snow cover, including thin first-year ice, ice structures on new ice called frost flowers, thick winter and spring ice, and surface melt ponds, despite exposure to high levels of potentially damaging radiation at the ice surface in summer. Most sea-ice bacteria derive from freezing seawater and inhabit the brine network within the ice, but bacteria delivered by atmospheric deposition are detected in overlying snow and surface melt ponds. Sea ice algae, especially diatoms, bloom in spring and summer in the brine channels of bottom ice, where they are bathed with seawater nutrients and receive sufficient sunlight; they have also been found in large aggregates at the bottom of melt ponds and as filamentous mats on the underside of the ice. The porous ice matrix and frost flowers are filled with extracellular polymeric substances (EPS), which are also involved in attachment of under-ice algal mats. (See [11] [18], and [19] for more detail). (B) Permafrost (not drawn to scale). In permanently frozen soil (grey), below the seasonally active layer (dark brown), bacteria and archaea can be found in abundance in cryopegs (buried lenses of relict seawater brines), where EPS concentrations are also high, and in veins of liquid brine that can exist between mineral grains. Freshwater ice wedges (white) that extend into permafrost also contain intact microorganisms, but at far lower abundances than in cryopegs or permafrost veins (See [13]and [14] for more detail).
Fig 2.
Glacial ecosystems showing simplified carbon cycle.
Photosynthetic and heterotrophic prokaryotes and eukaryotes inhabit the supraglacial ecosystem, which receives sunlight and has abundant liquid water. Organic matter is slowly transferred to englacial organisms and more rapidly transferred via hydrological processes to subglacial bacteria. The subglacial surface may be till or water, as in the case of subglacial lakes.
Fig 3.
Schematic and images of the compact, twin-beam digital holographic microscope.
(A) Schematic showing four main elements (discussed in the text): the source, the sample (specimen path is labeled Spec. and reference path is labeled Ref.), the microscope, and the sensor. (B) Solid model of the hardware. The fiber-fed source assembly is at the bottom, and the imaging camera is at the top. The microscope optics, comprised of the two aspheric lenses and the relay lens, are contained within the 300 mm long lens tube. In the laboratory, a three-axis stage between the source the microscope optics provides easy manual manipulation of the specimen under study. (C) Photograph of the field instrument (top case removed). The optical train, electronics, and computer are contained within a waterproof box. (D) Photo of instrument fully enclosed, as used in the field. The arrow indicates where a sample chamber is inserted; the structure pictured is a placeholder only.
Fig 4.
(A) Chambers had two PDMS channels, one for the reference beam and one for the object beam. (B) The chamber and plumbing fit into a cartridge with a gasket for sealing it into the instrument. Hand for scale. (C) Sample chamber being placed into the closed and sealed instrument.
Fig 5.
(A) Sackholes large enough to accommodate the instrument were drilled in sea ice with a Mark II ice corer to a depth of 20–25 cm, determined by first drilling test holes with an augur to determine ice thickness. (B) DHM in ice sackhole. (C) Sample from sackhole delivered to sample chamber via syringe. The box remains in the ice hole to keep the instrument equilibrated to in situ brine temperatures.
Fig 6.
Examples of eukaryotic cells and trajectories observed in sackhole brines of Malene Bay.
(A) Amplitude image of non-motile diatom. Note the clear demarcation of cell walls and organelles. (B, C) Rapidly swimming organism in amplitude (B) and phase (C) (the two images do not represent the same time point, but instead the best focus at the given z-plane, which differed in intensity vs. phase). (D) Minimum intensity amplitude projection giving the trajectory of the organism in (B,C) over 7 s. The arrow indicates the direction of motion. (E-G) More slowly swimming, typical organism seen in amplitude (E), phase (F), and the derivative of phase (G). (H) Trajectory of organism in (E-G) over 110 s (arrows). The cell reversed direction near the edge of the field. Note the trajectory of another, out-of-focus organism in the background (dashed arrows).
Fig 7.
Examples of prokaryotes and trajectories observed from Malene Bay seawater.
(A) Appearance of a nearly full-screen image containing objects suggestive of prokaryotes (arrows). (B) Zoomed-in appearance of a cell just out of the focal plane. (C) A cell at best focus. (D) Appearance of a prokaryote in a maximum intensity z-projection through 10 z planes (24 μm). The apparent size and contrast are increased. (E) Zig-zag motility of a prokaryote, observed as a maximum intensity projection through 60 s of time on a single z plane.
Fig 8.
Examples of trajectories of prokaryotic motility seen in Nuuk samples.
Images are maximum- or minimum-intensity projections across the length of time indicated in each image. (A) Malene Bay brine measured in situ with serine added to the sample chamber. (B) Kobbefjord brine measured in situ with serine added to the sample chamber. The ambient air temperature was –12°C. Note the non-motile organism for reference. (C) Another sample of Malene Bay brine measured in situ with serine added. (D) Malene Bay brine sample returned to the lab and stored at –4°C with measurement performed at –4°C. (E) Seawater sample warmed overnight to +4°C with the addition of half strength 2216 Marine Broth medium. (F) Brine sample warmed overnight to +4°C with the addition of half strength 2216 Marine Broth medium.
Fig 9.
Appearance of tracks of identified cells (prokaryotes and eukaryotes) in the presence of unequal serine concentrations.
The images are rotated 45° to reflect the orientation of the chamber in the microscope. (A) Serine introduced at the right edge of the sample chamber. (B) Serine introduced in the center top of the sample chamber. (C) Chemotaxis plot showing controls with drift and no serine gradient (green), cells from Panel A in blue, and cells from Panel B in red. Calculated x and y velocities of the cells are given in Table 1.
Table 1.
Total velocity, longitudinal (x) and transverse (y) velocities, and range of velocities seen for the samples pictured in Fig 9, as well as a control without serine.
Fig 10.
Images by epifluorescence microscopy of brine samples.
Organisms are stained with acridine orange and DAPI according to a dual-staining technique [41] and showed an assemblage of diatoms (A, C) and flagellates (B, D) from Malene Bay (A, B) and Kobbefjord (C, D). All scale bars are 10 μm. From Malene Bay: (A) A diatom, probably Navicula vanhoeffenii and (B) a flagellate, possibly Chlamydomonas or Telonemia; from Kobbefjord: (C) a diatom, probably Fragilariopsis oceanica, and (D) a flagellate, Pyraminmonas sp. (http://westerndiatoms.colorado.edu/) [46] [47].