Long-term Survival in Halite
Fluid inclusions in modern and ancient buried halite from Death Valley and Saline Valley, California, USA, contain an ecosystem of “salt-loving” (halophilic) prokaryotes and eukaryotes, some of which are alive. Prokaryotes may survive inside fluid inclusions for tens of thousands of years using carbon and other metabolites supplied by the trapped microbial community, most notably the single-celled alga Dunaliella, an important primary producer in hypersaline systems.
Reports of extreme microbe longevity in salt are controversial. The well-known Permian bacterium from the Waste Isolation Pilot Plant (WIPP) site, Salado Formation, New Mexico, USA (Vreeland et al., 2000), for example, comes from a brine inclusion within a large, diagenetically formed halite crystal. That brine inclusion could have been trapped after the Permian during burial cementation and recrystallization processes (Hazen and Roedder, 2001). Later study of those fluid inclusions, however, shows that they most likely contain evaporated Permian seawater, which supports their 250 Ma age and the antiquity of the trapped bacterium (Satterfield et al., 2005). The strongest criticism of the antiquity of prokaryotes recovered from ancient salt deposits has come from the biological science community, which maintains that deoxyribonucleic acid (DNA) should degrade over time scales far shorter than 250 m.y. in the absence of a repair mechanism (Willerslev et al., 2004; Hebsgaard et al., 2005; Willerslev and Hebsgaard, 2005). In addition, DNA from the Permian bacterium is nearly identical to a modern bacterium, Virgibacillus marismortui, sampled from the Dead Sea (Arahal et al., 1999, 2000), which suggests to some that the Permian bacterium is a laboratory contaminant (Graur and Pupko, 2001).
Halophilic Microorganisms in Modern Hypersaline Systems
The starting point for evaluating long-term survival of micro-organisms in fluid inclusions in salt is to examine modern evaporite systems and the processes by which organisms are preserved in halite there. We illustrate a typical hypersaline environment, Saline Valley, where, under certain conditions, surface brines host prolific numbers of halophilic microorganisms. Saline Valley is a closed-basin saline pan in eastern California that contains surface brines up to 0.5 m deep, fed by groundwaters (Figs. 1 and 2A) (Hardie, 1968; Howe, 1998). A bloom of planktonic halophiles, developed in March 2004, contained one type of photosynthetic autotroph, the single-celled alga Dunaliella, and many heterotrophs (prokaryotic Archaea and Bacteria, that thrived in bright red brines at salinities of 26%–30%, seven to eight and a half times more concentrated than seawater (Fig. 2B). The pink/red brine color is due to the carotenoids (organic pigments, including β-carotene used by microorganisms for protection from ultraviolet radiation) in halophilic Archaea and Bacteria and Dunaliella (Teller, 1987; Pedrós-Alió et al., 2000; Oren and Rodríguez-Valera, 2001; Oren, 2002b). Wet mounts prepared from Saline Valley brines contained rod- and coccoid-shaped prokaryotes and larger spherical and ellipsoid-shaped cells of Dunaliella, some of which were motile one year after collection (Figs. 2C and 2D).
Halophilic Microorganisms in Buried Pleistocene Salt
Borehole cores from Death Valley and Saline Valley, composed of interbedded salt and mud, provide ideal materials for assessing the fate of microbial communities trapped in fluid inclusions in halite in the subsurface for periods of up to 150 k.y. Fig. 4). The cored sediments contain a dated record of Pleistocene paleoenvironments that varied from saline pans and dry mudflats to deep, perennial lakes (Li et al., 1996; Howe, 1998; Lowenstein et al., 1999). Evaporites accumulated in two settings: (1) bedded halite with abundant primary growth textures formed in perennial saline lakes (i.e., Great Salt Lake, Utah, USA); and (2) massive halite formed in salt pans (i.e., Badwater Basin, Death Valley, USA) (Li et al., 1996; Lowenstein et al., 1999). Microorganisms in fluid inclusions were almost exclusively found in halites deposited in perennial saline lakes in Death Valley (ca. 10–35 ka) and Saline Valley (ca. 20 ka, 75 ka, and 150 ka). Some of these halites have prokaryotes in fluid inclusions comparable in abundance to those found in modern halites formed during the 2004 Saline Valley halophile bloom, which suggests that ancient saline lakes of Death Valley and Saline Valley were at times teeming with microorganisms (Schubert et al., 2009a).
Mechanism for Long-Term Survival of Prokaryotes in Fluid Inclusions
All Archaea from the Death Valley core we have cultured so far came from one stratigraphic interval (Fig. 4) in which prokaryotes and Dunaliella were observed in situ within fluid inclusions. Closer inspection of those fluid inclusions, coupled with what is known about the ecology of modern hypersaline systems, has led us to hypothesize a mechanism that may allow prokaryotes to survive inside fluid inclusions for millennia.
Although we are beginning to understand the community of microorganisms inside modern and ancient fluid inclusions, much more needs to be learned about how they survive. Miniaturized prokaryote cells suggest starvation-survival, despite the availability of carbon. We do not know why prokaryotes in fluid inclusions miniaturize, what factors trigger miniaturization, and what functions miniaturized cells are able to perform in fluid inclusions (e.g., repair of DNA and cell membranes) (Grant et al., 1998; Johnson et al., 2007). Alternatively, prokaryotes may form spores and survive for long periods in a dormant state, as has been claimed for the bacterium cultured from the Permian fluid inclusion by Vreeland et al. (2000). But none of the halophilic Archaea cultured from the Death Valley core formed endospores, nor do any Archaea. We thus need to learn more about long-term survival of spore-forming prokaryotes as well as miniaturized forms trapped in fluid inclusions. Such knowledge will be vital as studies further explore deep life on Earth and elsewhere in the solar system, where materials that potentially harbor microorganisms are millions and even billions of years old.