peak identities was further documented by analyzing several spectra (data not shown). Titanium has been found along with silicon coated on rock surfaces in the Ka’u desert on the Big Island [1.6] and in most Hawai’i steam cave/vent samples, but titanium has no known role in metabolism. The Lassen nonsulfur cave site SW1 was a shallow cave (Figure 1.2b) composed of soft backing material and the immediate sampling was guided by visual access. There was a distinctive red gelatinous material in the collected sample that proved to be noncellular upon microscopic examination. The nonsulfur SW1 spectrum (Figure 1.3b) had far fewer peaks than the Hawaiian H1 counterpart and contained as its main peaks silicon (Si) and oxygen (O) with smaller peaks for titanium (Ti) and carbon (C). Lassen iron vent SW4 was sampled as a thin (~2–3 mm) soft iron (III) oxide deposit collected from a hard rounded rock surface within the vent (Figure 1.2e, f). The spectrum (Figure 1.3c) contained strong peaks for iron (Fe), silicon (Si), oxygen (O) and aluminum (Al) with a moderate sulfur (S) peak and a smaller carbon (C) peak.
1.3.3 Steam and Steam Deposit Collection: Control Methods
Steam Controls
To control for the presence of foreign DNA in our sample analysis of DNA from steam-condensed samples, we used Millipore filtered (0.22 µm membrane) autoclaved Barnstead purified water. The water was converted to steam next to a cave or vent sampled and collected in identical fashion to steam samples from fumaroles. After collection, samples were analyzed with cave or vent samples by (1) PCR, (2) filtration of 10–15 mL samples on 13 mm black Millipore membranes (0.22 µm pore size) and DAPI microscopy, and (3) for thermophile culture pH 3 and 4.5 at 55, 70, 80, and 85 °C. Control samples proved to be negative.
Steam Deposit Controls
Steam deposit samples from Hawai’i H1 nonsulfur cave were collected by scraping lightly along the inside of the steam cave ceiling. Lava presents a hard surface that cannot be penetrated with a slight 50 mL polypropylene collection tube scraping able to collect steam deposits. We used an adjacent nonfumarolic, ambient temperature lava cave and performed an identical collection procedure with scraping-tube collection used for the steam cave. The other soft surfaces of Lassen nonsulfur cave SW1 and SW2 sulfur cave and Hawai’i H5 salt cave were shallow samplings only a few mm of thick deposits and did not include material other than from the nonsulfur steam deposits, salt deposits or sulfur sublimates. Steam deposit controls were analyzed and evaluated along with the collected steam cave/ vent samples, as described above for PCR, DAPI microscopy and thermophile culture and were negative. We are currently expanding our controls in several ways to evaluate the contribution growing cells might make to the chemical profile from EDX analysis.
Figure 1.3 Steam caves/vents spectra: (a) Hawai’i H1 nonsulfur cave. (b) Lassen nonsulfur cave SW1. (c) Lassen iron vent SW4. (Image credit: the authors).
1.4 Culture Isolation
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).
