Читать книгу EXTREMOPHILES as Astrobiological Models - Группа авторов - Страница 26

In some of the earliest work on low pH, high temperature organisms, the concept was advanced [1.5] that steam vents were lacking in microbial life and there was little encouragement to study any of those sites, since there were thousands of pools and springs available and already known. In steam caves/vents there is no mixing, settling or sediments as there might be in hot spring pools or flowing springs. Surfaces become an important part of the habitat since organisms must remain in place or be swept away from the habitat. As steam sweeps over these niche-habitats a continuous, though perhaps changing supply of nutrients passes through, some of which become concentrated or deposited as a nutrient source. Thus, the underside of ledges, ceilings of caves, or sides of vents all become important areas for colonization and consequently, for collection. In this regard, we collected from a steam cave ledge, but only on the underside where steam rose up and hit the ledge deep inside a cave, or on the salt cave shelf where pure white salt crystals existed (Figure 1.2d) and could be collected. When we collected from narrow caves on the ceiling often it was simply a hard ceiling so that no soil or other features existed beyond the hard lava ceiling and the steam deposits on the ceiling structure, sometimes several meters inside a horizontal cave.

Typically, steam deposit samples collected from caves and vents were enriched in vials in mineral salts medium [1.8]. A small amount of a natural sample was aseptically added to a vial containing 10 mL liquid medium at temperatures in stepwise increases. At lower temperatures (55–75 °C), Teflon caps were used; at higher temperatures removable screw cap butyl stoppers replaced these. We established enrichments in the present study with three high temperature sites in Lassen Volcanic National Park, a nonsulfur cave SW1, a sulfur cave SW3 and an iron vent SW4. Cultures set at pH 4.5, 85 °C were positive in 10–15 days. All of the cultures that were positive were slightly below the habitat temperature. When cultures were examined microscopically there was commonly a limited variety of organisms ranging from irregular spheres to very thin rods and irregular spheres in a mixed culture usually with thin filaments. Examples of DAPI stained cultures appear in Figures 1.4 to 1.6. All the SW1 and SW4 cultures were initially isolated at pH 4.5, 80 °C, but with subculture one of them, SW4, grew more densely at pH 3, 80 °C, its habitat pH, 6.50 × 10⁸ cells mL^-1 compared to 1.48 × 10⁸ cells mL^-1 at pH 4.5, 80 °C. Large cell groups consisting of hundreds of cells were seen throughout the iron vent cultures growing at pH 4.5 and 80 °C (Figure 1.6).

Figure 1.4 Sulphur Works nonsulfur cave SW1 culture pH 4.5, 80 °C. (a) Phase contrast, numerous cell pairs and single cells appear phase dense. (b) DAPI stain. Cells stained by DAPI appear blue. Cell pairs often appear as a result of cell division. (c) Phase contrast. Large cell groups appear in culture associated with reddish tinged cave deposit material (arrowhead). Phase dense cells appear spherical. (d) DAPI stain. Reddish tinge minerals (arrowhead) and associated cells at the surface adhering to mineral particles. Bar, A–D, 10 µm. (Image credit: the authors).

Figure 1.5 Sulphur Works sulfur cave SW3 culture pH 4.5, 85 °C. (a) Phase contrast (b) DAPI stain. Mixed culture; spheres identified by PCR as Sulfolobus acidocaldarius and thin filaments, identity unknown. Microscopic field with approximately equal numbers of spheres and thin filaments. Bar A–B, 10 µm. (Image credit: the authors).

The appearance of the irregular spheres isolated from SW1 and SW4 resembled Archaea, but more information was essential to determine their taxonomic status. In some instances, particularly where heavy metals may be detrimental to DNA integrity, it may not be possible to obtain amplifiable DNA, or if DNA is obtained it may not sequence properly. If it is not possible to obtain DNA for identification from cultures, fluorescence in situ hybridization (FISH) is used to identify the taxonomic group. An example of this approach is given in Figure 1.7. The controls include DAPI stain, to identify Bacteria and Archaea, a nonsense sequence probe (NON338) and no probe added. Both samples and controls were processed identically and at the same time. Negative results were obtained with the controls and indicated that if the Archaea-specific Arch915 probe labeled cells, the labeling when positive indicated the cells were Archaea. In this sample from a culture of mixed rods and spheres only the spheres were labeled by the probe, indicating that they were members of the taxonomic group Archaea, although no further information on their taxonomy is available from this direct labeling technique.

Figure 1.6 Sulphur Works iron vent SW4 culture, pH 4.5, 80 °C. (a) Phase contrast. Silica, iron and minerals form a heterogeneous base for cell growth in culture. Arrowheads (a,b) indicate same cell location. (b) DAPI stain. Cells appear as blue fluorescent spheres mainly surrounding edges of steam deposits. (c) Phase contrast. Faint red tinged siliceous particle with few cells seen, other than at lower right edge. (d) DAPI stain. Same small cell group evident at lower right edge in phase contrast and DAPI images; blue DAPI stained cells, not seen by phase contrast, appear dispersed throughout the deposit particle. Bar A–D, 5 µm. (Image credit: the authors).

Figure 1.7 FISH labeled culture pH 4.5, 80 °C, isolated from iron vent, Sulphur Works SW4. (a) Phase contrast. (b) DAPI stain. (c) Fluorescent image of cells labeled with Archaea-specific probe Arch915-CY3 probe. Labeled cells were all bright orange archaeal spheres. None of the rod-shaped bacterial cells (A,B, arrowhead) were labeled by the probe (c). Only spherical Archaea were fluorescent. Arrowheads (a-c) mark location of a typical bacterial rod nonlabeled in C. Bar A–C, 5 µm. (Image credit: the authors).

A salt cave liquid enrichment, Hawai’i H5, was obtained at pH 4.5, 55 °C. The culture was a mixture of thin filaments, wide rods about 0.5 µm in diameter, and pleomorphic cells. Further work with more selective conditions (ionic species, pH, temperature) and solid surface isolations may assist in obtaining pure cultures of the organisms present.