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For sampling steam deposit samples when steam caves are too hot for handheld sampling tools, or samples are collected deep within a cave interior, an extension pole is used with a sterile 50 mL polypropylene tube angled up to 45° towards the cave opening. With this approach, only the upper trailing edge of the open tube contacts the deposit matrix. Thus, the steam deposit material removed by the upper collection tube edge falls directly into the open tube, which is capped immediately upon collection. In most fumaroles, collected material is visible and only a few mm (2–4 mm) at the steam-cave surface deposit is removed. Most collection sites have either thick or easily removed deposits of sulfur, salt, iron or other matrix material, making this procedure a relatively easy task. In cases where the steam contact site cannot be seen, the surface is lava, so only a light sampling is carried out, just sufficient to remove the adherent steam deposit material.

Hawai’i presented the greatest variety of chemical types of steam vents. Three of the vents, two coded and one noncoded, were nonsulfur vents. This type of vent is recharged by meteoric input. Rainwater descends through porous ground, contacts rising heat and is converted to steam. Ammonia released by degassing magma creates an ascending mixture of ionized and NH₃ gas traveling upward through fissures and fractured lava, reaching passage-ways that lead to horizontal or vertical caves and vents to the atmosphere as fumaroles. In Hawai’i these clouds of rising steam vapors containing volcanic gases and ionized elements support the growth of ammonia oxidizing archaea (AOA) located below the vent opening and adhering to the cave ceilings and walls. Ammonia oxidizing archaea gain their nutrients from entrapped particles, condensing steam and concentrating volcanic gases such as ammonia that rise from closely positioned magma that degases as it rises towards the surface. Altogether these events in the Hawaiian nonsulfur caves support an unusual habitat that thrives on the concentrating effects of surfaces with matrix material and contact between the steam-gas mixture. Here, the cooler cave ceiling or surface condenses steam from the continuous flow of warm thermal vapors and nutrients. Figure 1.2 shows two nonsulfur sites, one in Hawai’i and one in Lassen Volcanic National Park along with a sulfur cave and an iron vent in Lassen, and a salt cave site in Hawai’i Volcanoes National Park. We have documented the presence of AOA in three nonsulfur steam vents in Hawai’i and have recently isolated Archaea-like cultures from the nonsulfur cave and Archaea from the iron vent in Lassen Park. We have not yet determined whether the SW1 nonsulfur cave Archaea-like spheres belong to the Thaumarchaeota, a newly proposed phylum for the ammonia oxidizing archaea [1.4] [1.14] [1.15]. The Hawai’i H1 steam seems similar to the steam at the nonsulfur vent in Lassen Volcanic National Park (Table 1.2) and there is sufficient ammonia at both sites for AOA, and clones were recovered at the Hawai’i H1 site [1.1].

We recognized that a majority of microorganisms growing in natural habitats attach to surfaces. Since there is no sediment in steam caves, organisms tend to attach to the type of surface deposit created by the steam flow and chemical identity of the steam vent or cave (Table 1.1). The chemical features of solid surfaces can be characterized by X-ray microanalysis. As a result, steam deposit samples from caves and vents can be analyzed directly for their chemical composition. In our case, we used an X-max 50 mm2 X-ray detector and a Quanta 450 FEI-SEM operated at 20 kV with Oxford Inca software. Samples were attached with an adhesive-conductive carbon tab to a stub and analyzed. We collected spectra (Figure 1.3) from three different types of caves/vents that supported growth:, (a) Hawai’i H1 nonsulfur cave, (b) Lassen SW1 nonsulfur cave, (c) Lassen SW4 iron vent and Hawai’i H5 salt cave. The salt cave sample remains to be analyzed. The Hawaiian sites H1 and H5 were 65–68 °C and the Lassen sites SW1 and SW4 were 85.5–87 °C.

Figure 1.2 Steam deposit sampling sites: (a) Nonsulfur steam cave site Hawai’i 1, rising steam vapors fill the air to the left of the cave opening (arrow). Ammonia oxidizing archaea (AOA) clones recovered from H1. (b) Nonsulfur steam cave site SW1 Lassen, unknown spherical cell culture recovered and subcultured. (c) Sulfur steam cave SW3 Lassen, steam vapor clouds mix with air and deposit yellow S° inside cave and on outside rock surfaces. Sulfolobus cultures recovered from SW3 and subcultured, pH 4.5, 85 °C. (d) Salt cave site Hawai’i 5, rainwater-lava interaction result in salt cave deposits (white) on the ceiling, wall and floor deep within Hawai’i 5. Mixed culture enrichments, pH 4.5, 55 °C, include abundant unknown thin filaments. (e) Iron steam vent site SW4 Lassen, unknown archaeon recovered and subcultured. (f) Measuring temperature in iron vent SW4 with mercury maximum recording thermometer (arrow). B, E Cultures isolated and subcultured at pH 4.5, 80 °C; SW4 isolate was also subcultured at pH 3, 80 °C. (Image credit: the authors).

Table 1.2 Analysis of steam deposits in Hawai’i and Lassen fumaroles.¹

Analyte (mg L-1)	H1NS°C2	SW1NS°C	SW4FeV3
Na	3.454	3.457	3.453
Ca	2.285	2.119	2.870
Al	1.024	1.258	2.006
Fe (total)	0.975	0.974	0.997
Si	18.579	21.932	25.706
B	0.697	0.723	0.760
K	2.864	3.965	3.401
Mg	0.119	0.138	0.945
Zn	0.0147	0.00455	0.0362
Mn	0.00604	0.00618	0.0256
Mo	0.00	0.00	0.00
Se	0.0289	0.0242	0.0251
Ni	0.00	0.00	0.00
Pb	0.00	0.00	0.00
Cr	0.00	0.00	0.00
Cd	0.00164	0.00178	0.00236
Cu	0.00	0.00	0.00
Hg	0.00	0.00	0.00
As (total)	0.0242	0.0201	0.0209
S	0.0312	5.143	17.833
Sr	0.0832	0.00596	0.0187
NO2/NO3, N-NO3 (µM)	0.00	0.00	3.453
NH4,N-NH4 (µM)	19.550	79.023	5.780
PO4,P-PO4 (µM)	0.00	27.372	0.00
SO4 (µM)	0.00	0.00	222.0
Conductivity (µScm-1)	13.9	167.2	516

¹ Analysis by inductively coupled plasma optical emission spectrometry and wet chemistry.

² Column 1 Data from Bizzoco and Kelley [1.3].

³ Column 3 data from Bizzoco and Kelley [1.2].

H1NS°C, Hawai’i 1 nonsulfur cave, SW1NS°C Sulphur Works 1 nonsulfur cave, SW4FeV, Sulphur Works 4 iron vent.

The Hawai’i H1 sample (Figure 1.3a) was collected from a hard lava cave ceiling (Figure 1.2a) and contained strong peaks for aluminum (Al), silicon (Si) and iron (Fe). The aluminum peak was also formed by a smaller peak of iridium (Ir). Although characterized as a nonsulfur cave, a small sulfur (S) peak was present in the collected sample and was a characteristic of this cave site. Also observed was a strong peak for oxygen (O) and moderate peaks for titanium (Ti) and several biologically important elements, potassium (K), sodium (Na), magnesium (Mg) and calcium (Ca); each of these 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.