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Recent studies claiming to revive ancient microorganisms trapped in fluid inclusions in halite have warranted an investigation of long-term microbial persistence. While starvation-survival is widely reported for bacteria, it is less well known for halophilic archaea—microorganisms likely to be trapped in ancient salt crystals. To better understand microbial survival in fluid inclusions in ancient evaporites, laboratory experiments were designed to simulate growth of halophilic archaea under media-rich conditions, complete nutrient deprivation, and a controlled substrate condition (glycerol-rich) and record their responses. Haloarchaea used for this work included Hbt. salinarum and isolate DV582A-1 (genus Haloterrigena) sub-cultured from 34 kyear Death Valley salt. Hbt. salinarum and DV582A-1 reacted to nutrient limitation with morphological and population changes. Starved populations increased and most cells converted from rods to small cocci within 56 days of nutrient deprivation. The exact timing of starvation adaptations and the physical transformations differed between species, populations of the same species, and cells of the same population. This is the first study to report the timing of starvation strategies for Hbt. salinarum and DV582A-1. The morphological states in these experiments may allow differentiation between cells trapped with adequate nutrients (represented here by early stages in nutrient-rich media) from cells trapped without nutrients (represented here by experimental starvation) in ancient salt. The hypothesis that glycerol, leaked from Dunaliella, provides nutrients for the survival of haloarchaea trapped in fluid inclusions in ancient halite, is also tested. Hbt. salinarum and DV582A-1 were exposed to a mixture of lysed and intact Dunaliella for 56 days. The ability of these organisms to utilize glycerol from Dunaliella cells was assessed by documenting population growth, cell length, and cell morphology. Hbt. salinarum and DV582A-1 experienced size reductions and shape transitions from rods to cocci. In the short-term, these trends more closely resembled the response of these organisms to starvation conditions than to nutrient-rich media. Results from this experiment reproduced the physical state of cells (small cocci) in ancient halite where prokaryotes co-exist with single-celled algae. We conclude that glycerol is not the limiting factor in the survival of haloarchaea for thousands of years in fluid inclusions 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. Deeper understanding of the long-term survival of prokaryotes in fluid inclusions will complement studies that further explore microbial life on Earth and elsewhere in the solar system, where materials that potentially harbor microorganisms are millions and even billions of years old.

A 90-m-long (100,000 year old) salt core from Death Valley, California, contains cells of the algal genus Dunaliella co-trapped with prokaryote cells in fluid inclusions in halite. It is hypothesized that Dunaliella cells provided glycerol, the carbon source needed by halophilic Archaea for survival over periods of tens of thousands of years. Support for this hypothesis includes: observations that intracellular materials leaked from Dunaliella cells into fluid inclusions; the distribution of Dunaliella cells in the Death Valley core, which matches the distribution of culturable prokaryotic cells; and halophilic Archaea cultured from the Death Valley core grew in media containing glycerol as the only carbon source.

Long-term survival of microorganisms has been demonstrated by prokaryotes cultured from ancient halite, but previous results are controversial. Three genera of non-spore-forming halophilic Archaea were cultured from 22–34 k.y. old subsurface halite from Death Valley, California. Primary, brine-filled inclusions in this halite contained prokaryotic organisms in miniaturized starvation-survival forms and dead cells of the algal genus Dunaliella . The energy needed for protracted survival of halophilic Archaea , including repair of damaged DNA, may have been provided by glycerol and other carbon molecules leaked from Dunaliella cells. These results provide further evidence that fluid inclusions in halite are a favorable refuge for long-term survival of microorganisms, and indicate that the original depositional environment influences the distribution and viability of prokaryotes.

Bacteria and archaea isolated from crystals of halite 104 to 108 years old suggest long-term survival of halophilic microorganisms, but the results are controversial. Independent verification of the authenticity of reputed living prokaryotes in ancient salt is required because of the high potential for environmental and laboratory contamination. Low success rates of prokaryote cultivation from ancient halite, however, hamper direct replication experiments. In such cases, culture-independent approaches that use the polymerase chain reaction (PCR) and sequencing of 16S ribosomal DNA are a robust alternative. Here, we use amplification, cloning, and sequencing of 16S ribosomal DNA to investigate the authenticity of halophilic archaea cultured from subsurface halite, Death Valley, California, 22,000 to 34,000 years old. We recovered 16S ribosomal DNA sequences that are identical, or nearly so (>99%), to two strains, Natronomonas DV462A and Halorubrum DV427, which were previously isolated from the same halite interval. These results provide the best independent support to date for the long-term survival of halophilic archaea in ancient halite. PCR-based approaches are sensitive to small amounts of DNA and could allow investigation of even older halites, 106 to 108 years old, from which microbial cultures have been reported. Such studies of microbial life in ancient salt are particularly important as we search for microbial signatures in similar deposits on Mars and elsewhere in the Solar System. Key Words: Ancient DNA—Halite—Haloarchaea—Long-term survival. Astrobiology 14, 553–560.

Fluid inclusions in evaporite minerals (halite, gypsum, etc.) potentially preserve genetic records of microbial diversity and changing environmental conditions of Earth's hydrosphere for nearly one billion years. Here we describe a robust protocol for surface sterilization and retrieval of DNA from fluid inclusions in halite that, unlike previously published methods, guarantees removal of potentially contaminating surface-bound DNA. The protocol involves microscopic visualization of cell structures, deliberate surface contamination followed by surface sterilization with acid and bleach washes, and DNA extraction using Amicon centrifugal filters. Methods were verified on halite crystals of four different ages from Saline Valley, California (modern, 36 ka, 64 ka, and 150 ka), with retrieval of algal and archaeal DNA, and characterization of the algal community using ITS1 sequences. The protocol we developed opens up new avenues for study of ancient microbial ecosystems in fluid inclusions, understanding microbial evolution across geological time, and investigating the antiquity of life on earth and other parts of the solar system.

Systematic changes in the chemistry of evaporated seawater contained in primary fluid inclusions in marine halites indicate that seawater chemistry has fluctuated during the Phanerozoic. The fluctuations are in phase with oscillations in seafloor spreading rates, volcanism, global sea level, and the primary mineralogies of marine limestones and evaporites. The data suggest that seawater had high Mg 2+ /Ca 2+ ratios (>2.5) and relatively high Na + concentrations during the Late Precambrian [544 to 543 million years ago (Ma)], Permian (258 to 251 Ma), and Tertiary through the present (40 to 0 Ma), when aragonite and MgSO 4 salts were the dominant marine precipitates. Conversely, seawater had low Mg 2+ /Ca 2+ ratios (+ concentrations during the Cambrian (540 to 520 Ma), Silurian (440 to 418 Ma), and Cretaceous (124 to 94 Ma), when calcite was the dominant nonskeletal carbonate and K-, Mg-, and Ca-bearing chloride salts, were the only potash evaporites.

CaCl 2 basinal brines, which are present in most Phanerozoic sedimentary basins, inherited their chemistries and salinities from evaporated paleoseawaters when the world oceans were Ca rich and SO 4 poor (CaCl 2 seas). CaCl 2 seas coincided with periods of rapid seafloor spreading, high influxes of mid-ocean-ridge brines rich in CaCl 2 , and elevated sea levels, conditions that favored accumulation of marine CaCl 2 brines in marginal and interior continental basins. Typical basinal brines in Silurian-Devonian formations of the interior Illinois basin, United States, show the same compositional trends as those of progressively evaporated CaCl 2 -rich Silurian seawater. Chemical deviations can be accounted for quantitatively by brine-rock reactions during burial (dolomitization, dolomite and K-feldspar cement). This explanation for the origin of CaCl 2 basinal brines contrasts with others that assume constancy of seawater chemistry and involve more complex brine-rock interactions.