Bacterial Activity at -2 to -20 degrees C in Arctic wintertime sea ice

Abstract

Arctic wintertime sea-ice cores, characterized by a temperature gradient of -2 to -20 degrees C, were investigated to better understand constraints on bacterial abundance, activity, and diversity at subzero temperatures. With the fluorescent stains 4',6'-diamidino-2-phenylindole 2HCl (DAPI) (for DNA) and 5-cyano-2,3-ditoyl tetrazolium chloride (CTC) (for O(2)-based respiration), the abundances of total, particle-associated (>3- micro m), free-living, and actively respiring bacteria were determined for ice-core samples melted at their in situ temperatures (-2 to -20 degrees C) and at the corresponding salinities of their brine inclusions (38 to 209 ppt). Fluorescence in situ hybridization was applied to determine the proportions of Bacteria, Cytophaga-Flavobacteria-Bacteroides (CFB), and Archaea. Microtome-prepared ice sections also were examined microscopically under in situ conditions to evaluate bacterial abundance (by DAPI staining) and particle associations within the brine-inclusion network of the ice. For both melted and intact ice sections, more than 50% of cells were found to be associated with particles or surfaces (sediment grains, detritus, and ice-crystal boundaries). CTC-active bacteria (0.5 to 4% of the total) and cells detectable by rRNA probes (18 to 86% of the total) were found in all ice samples, including the coldest (-20 degrees C), where virtually all active cells were particle associated. The percentage of active bacteria associated with particles increased with decreasing temperature, as did the percentages of CFB (16 to 82% of Bacteria) and Archaea (0.0 to 3.4% of total cells). These results, combined with correlation analyses between bacterial variables and measures of particulate matter in the ice as well as the increase in CFB at lower temperatures, confirm the importance of particle or surface association to bacterial activity at subzero temperatures. Measuring activity down to -20 degrees C adds to the concept that liquid inclusions in frozen environments provide an adequate habitat for active microbial populations on Earth and possibly elsewhere.

FIG. 1.

Microscopic images of wintertime sea ice from the Chukchi sea near Barrow, Alaska, at −5 (A) and −15°C (B). Ice-grain boundaries and triple-point junctures (upper panels) and details of brine pockets (lower left panels, which are enlargements of the areas boxed in red in the upper panels) are visible by transmitted light. DAPI-stained bacteria (blue) attached to the wall of a brine pocket (A) or to particulate material within the pocket (B) are visible in the same fields as those shown in the lower left panels when examined by epifluorescence light (lower right panels).

FIG. 2.

Microscopic images of wintertime sea ice at −20°C. The lower left panel is an enlargement of the area boxed in red in the upper panel. The images are similar to those in Fig. 1, except that a triple-point juncture is not obvious and the DAPI-stained bacterium (lower right panel) is not attached to a surface.

FIG. 3.

Fractions of total bacteria that were attached (A) or active (B) and fractions of active cells that were attached (C) across the temperature gradient in wintertime sea ice. Circles indicate data from intact ice sections examined microscopically. Diamonds indicate mean values from isothermal-isohaline-melted ice samples used for CTC incubations: black diamonds indicate samples from the Chukchi Sea, and gray diamonds indicate samples from Elson Lagoon. Triangles indicate mean values from isothermal-isohaline-melted ice samples used for rRNA probing. Error bars indicate the SEM (n = 3).

FIG. 4.

Images obtained by epifluorescence microscopy of particle-associated bacteria in isohaline-isothermal-melted samples of wintertime ice from the Chukchi Sea at −20°C. Hybridization with fluorescent probes is shown for Bacteria (A) and Archaea (B).

FIG. 5.

Fractions of Bacteria that were CFB (A) and fractions of total cells that were Archaea (B) across the temperature gradient in wintertime sea ice. Error bars indicate the SEM (n = 3).