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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.

Summary Halophilic Archaea cultured from ancient fluid inclusions in a 90-m-long (0- to 100 000-year-old) salt core from Death Valley, California, demonstrate survival of bacterial cells in subsurface halite for up to 34 000 years. Five enrichment cultures, representing three genera of halophilic Archaea (Halorubrum, Natronomonas and Haloterrigena), were obtained from five surface-sterilized halite crystals exclusively in one section of the core (13.0–17.8 m; 22 000–34 000 years old) containing perennial saline lake deposits. Prokaryote cells were observed microscopically in situ within fluid inclusions from every layer that produced culturable cells. Another 876 crystals analysed from depths of 8.1–86.7 m (10 000–100 000 years old) failed to yield live halophilic Archaea. Considering the number of halite crystals tested (culturing success of 0.6%), microbial survival in fluid inclusions in halite is rare and related to the paleoenvironment, which controls the distribution and abundance of trapped microorganisms. Two cultures from two crystals at 17.8 m that yielded identical 16S rRNA sequences (genus: Haloterrigena) demonstrate intra-laboratory reproducibility. Inter-laboratory reproducibility is shown by two halophilic Archaea (genus: Natronomonas), with 99.3% similarity of 16S rRNA sequences, cultured from the same core interval, but at separate laboratories.

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.

DNA was extracted from surface-sterilized salt of different geological ages (23, 121, 419 million years of age, MYA) to investigate haloarchaeal diversity. Only Haloarcula and Halorubrum DNA was found in 23 MYA salt. Older crystals contained unclassified groups and Halobacterium. The older crystals yielded a unique 55-bp insert within the 16S rRNA V2 region. The secondary structure of the V2 region completely differed from that in haloarchaea of modern environments. The DNA demonstrates that unknown haloarchaea and the Halobacterium were key components in ancient hypersaline environments. Halorubrum and Haloarcula appear to be a dominant group in relatively modern hypersaline habitats.

Primary fluid inclusions in halite crystallized in Saline Valley, California, in 1980, 2004-2005, and 2007, contain rod- and coccoid-shaped microparticles the same size and morphology as archaea and bacteria living in modern brines. Primary fluid inclusions from a well-dated (0-100,000 years), 90 m long salt core from Badwater Basin, Death Valley, California, also contain microparticles, here interpreted as halophilic and halotolerant prokaryotes. Prokaryotes are distinguished from crystals on the basis of morphology, optical properties (birefringence), and uniformity of size. Electron micrographs of microparticles from filtered modern brine (Saline Valley), dissolved modern halite crystals (Saline Valley), and dissolved ancient halite crystals (Death Valley) support in situ microscopic observations that prokaryotes are present in fluid inclusions in ancient halite. In the Death Valley salt core, prokaryotes in fluid inclusions occur almost exclusively in halite precipitated in perennial saline lakes 10,000 to 35,000 years ago. This suggests that trapping and preservation of prokaryotes in fluid inclusions is influenced by the surface environment in which the halite originally precipitated. In all cases, prokaryotes in fluid inclusions in halite from the Death Valley salt core are miniaturized (

The major-ion (Mg2+, Ca2+, Na+, K+, SO 4 2 - , and Cl−) chemistry of Cretaceous seawater was determined from analyses of seawater-derived brines preserved as fluid inclusions in marine halites. Fluid inclusions in primary halite from three evaporite deposits were analyzed by the environmental scanning electron microscopy (ESEM) X-ray energy dispersive spectrometry (EDS) technique: the Early Cretaceous (Aptian, 121.0–112.2 Ma) of the Sergipe basin, Brazil and the Congo basin, Republic of the Congo, and the Early to Late Cretaceous (Albian to Cenomanian, 112.2–93.5 Ma) of the Khorat Plateau, Laos, and Thailand. The fluid inclusions in halite indicate that Cretaceous seawater was enriched several fold in Ca2+, depleted in SO 4 2 - , Na+, and Mg2+, and had lower Na+/Cl−, Mg2+/Ca2+, and Mg2+/K+ ratios compared to modern seawater. Elevated Ca2+ concentrations, with Ca2+ > SO 4 2 - at the point of gypsum saturation, allowed Cretaceous seawater to evolve into Mg2+–Ca2+–Na+–K+–Cl− brines lacking measurable SO 4 2 - .The major-ion composition of Cretaceous seawater was modeled from fluid inclusion chemistries for the Aptian and the Albian-Cenomanian. Aptian seawater was extreme in its Ca2+ enrichment, more than three times higher than present day seawater, with a Mg2+/Ca2+ ratio of 1.1–1.3. Younger, Albian-Cenomanian seawater had lower Ca2+ concentrations, and a higher Mg2+/Ca2+ ratio of 1.2–1.7. Cretaceous (Aptian) seawater has the lowest Mg2+/Ca2+ ratios so far documented in Phanerozoic seawater from fluid inclusions in halite, and within the range chemically favorable for precipitation of low-Mg calcite ooids and cements. Results from halite fluid inclusions, together with Mg2+/Ca2+ ratios measured from echinoderm and rudist calcite, all indicate that Early Cretaceous seawater (Hauterivian, Barremian, Aptian, and Albian) had lower Mg2+/Ca2+ ratios than Late Cretaceous seawater (Coniacian, Santonian, and Campanian). Low Aptian-Albian Mg2+/Ca2+ seawater ratios coincide with negative excursions of 87Sr/86Sr ratios and δ34SSO4, and peak Cretaceous ocean crust production rates, all of which suggests a link between seawater chemistry and midocean ridge hydrothermal brine flux.

The major-ion (Mg2+, Ca2+, Na+, K+, SO42−, and Cl−) composition of Permian seawater was determined from chemical analyses of fluid inclusions in marine halites. New data from the Upper Permian San Andres Formation of Texas (274–272 Ma) and Salado Formation of New Mexico (251 Ma), analyzed by the environmental scanning electron microscopy (ESEM) X-ray energy-dispersive spectrometry (EDS) method, along with published chemical compositions of fluid inclusions in Permian marine halites from North America (two formations of different ages) and the Central and Eastern European basins (eight formations of four different ages) show that Permian seawater shares chemical characteristics with modern seawater, including SO42− > Ca2+ at the point of gypsum precipitation, evolution into Mg2+-Na+-K+-SO42−-Cl− brines, and Mg2+/K+ ratios ∼5. Permian seawater, however, is slightly depleted in SO42− and enriched in Ca2+, although modeling results do not rule out Ca2+ concentrations close to those in present-day seawater. Na+ and Mg2+ in Permian seawater are close to (slightly below) their concentrations in modern seawater. Permian and modern seawater are both classified as aragonite seas, with Mg2+/Ca2+ ratios >2, conditions favorable for precipitation of aragonite and magnesian calcite as ooids and cements. The chemistry of Permian seawater was modeled using the chemical composition of brine inclusions for three periods: Lower Permian Asselian-Sakmarian (296–283 Ma), Lower Permian Artinskian-Kungurian (283–274 Ma), and Upper Permian Tatarian (258–251 Ma). Parallel changes in the chemistry of brine inclusions from equivalent age evaporites in North America, Central Europe, and Eastern Europe show that seawater underwent secular variations in chemistry over the 50 million years of the Permian. Modeled SO42− concentrations are 20 mmol per kg H2O (mmolal) and 19 mmolal in the Asselian-Sakmarian and Artinskian-Kungurian, with higher concentrations in the Upper Permian Tatarian (23 mmolal). Modeled Ca2+ is at or above its concentration in modern seawater throughout the Permian. Mg2+ is close to (slightly below) its concentration in modern seawater (55 mmolal) in the Asselian-Sakmarian (52 mmolal), and Tatarian (52 mmolal), but slightly higher than modern seawater in the Artinskian-Kungurian (60 mmolal). Mg2+/Ca2+ ratios are 3.5 (total range = 2.7 to 5.5) in the Lower Permian and rose slightly to 3.7 (total range = 3.1 to 5.8) in the Upper Permian, primarily due to decreases in Ca2+. These results are consistent with models that predict oscillations in the major-ion composition of Phanerozoic seawater on the basis of changes in the midocean ridge/river water flux ratio driven by changes in the rate of midocean ridge crust production. The Permian was characterized by low sea levels, icehouse conditions, and southern hemisphere glaciation. Such conditions, analogous to the present ice age, and the similarities between Permian seawater and modern seawater, all suggest that general Phanerozoic supercycles, driven by mantle convection and global volcanicity, also control the major-ion chemistry of seawater.

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 from marine halites have long been studied to determine the chemical composition of ancient seawater. Chemical analyses of the major ions in fluid inclusions in halites from the solar saltwork of Great Inagua Island, Bahamas, and from the supratidal sabkha, Baja California, Mexico, show that modern marine halites faithfully record the chemical signature of seawater. The major ions in Great Inagua and Baja California fluid inclusions display distinctive linear trends when plotted against one another (ie., Na+, K+, and SO42− vs. Mg2+ and Cl−), which track the evaporation path of seawater as it evolved during the crystallization of halite. These evaporation paths defined for the major ions by fluid inclusions in halite overlap findings of computer simulations of the evaporation of modern seawater by the Harvie, Moller, and Weare (HMW) computer program. The close match between the HMW seawater evaporation paths and the Great Inagua fluid inclusion data is not surprising considering the carefully controlled inflow, evaporation, and discharge of seawater at the Great Inagua saltwork. The major ion chemistry of fluid inclusions from the Baja California halites matches the HMW seawater evaporation paths in most respects, but one Baja fluid inclusion has lower concentrations of Mg2+ than evaporated seawater. Nonmarine inflows and syndepositional recycling of preexisting salts in the Baja California supratidal setting were not large enough to override the chemical signature of evaporating seawater as the primary control on the Baja fluid inclusion compositions. Fluid inclusions in halites from the nonmarine Qaidam Basin, Qinghai Province, western China, have a distinctly different major ion chemical signature than does “global” seawater. The fluid inclusion chemistries from the Qaidam Basin halites do not lie on the evaporation pathways defined by modern seawater and can clearly be differentiated from fluid inclusions containing evaporated seawater. If fluid inclusions in halites from modern natural settings contain unmistakable samples of evaporated seawater, then evaluation of the chemistry of ancient seawater by chemical analysis of fluid inclusions in ancient marine halites by means of the same approach should be valid.

Quantitative chemical analyses of fluid inclusions in sedimentary and diagenetic minerals can greatly improve our understanding of the chemistry of ancient surface and near surface waters. The environmental scanning electron microscope (ESEM) with an attached X-ray energy dispersive system (EDS) is capable of producing rapid and accurate major element chemical analyses of individual fluid inclusions in crystals of halite greater than about 30 μm in diameter. The ESEM-EDS technique uses the same basic principles as the X-ray microanalysis of frozen fluid inclusions reported by Ayora and Fontarnau [Ayora, C., Fontarnau, R., 1990. X-ray microanalysis of frozen fluid inclusion. Chemical Geology, 89, pp. 135–148.] but operates in a low-vacuum environment, which allows direct observation of the fluid inclusions to be analyzed without the need for a conductive coating. Modifications of the technique of Ayora and Fontarnau [Ayora, C., Fontarnau, R., 1990. X-ray microanalysis of frozen fluid inclusion. Chemical Geology, 89, pp. 135–148.] also include a newly designed sample holder, modified methods for the preparation of standard brine solutions to obtain “glass-like” homogeneous freezing behavior by addition of ethylene glycol, and a technique to obtain flat smooth surfaces on standard brine solutions. The ability to observe samples before analysis led to the discovery of heterogeneous freezing behavior of some standard solutions and fluid inclusions, which adversely influenced measurement reproducibility. The ESEM-EDS technique yielded quantitative results for Ca, Mg, K, SO 4 , and Cl at concentrations above about 0.1 wt.%; for Na, quantitative analyses were achieved at concentrations above 0.5 wt.%. Accuracies for the major elements in aqueous standards were better than 7% and precisions (relative standard deviations) ranged from 2% to 7%. Results on fluid inclusions in laboratory-grown halite gave accuracies of 6% to 10% for Mg, Ca, and K and precisions from 3% to 16%.

Carotenoids are common components of many photosynthetic organisms and are well known from the red waters of hypersaline ecosystems where they are produced by halophilic algae and prokaryotes. They are also of great interest as biomarkers in extraterrestrial samples. Few laser Raman spectroscopy studies have examined ancient field samples, where pigments and microscopic life are less defined. Here, we have identified carotenoids in ancient halite brine inclusions, 9 ka to 1.44 Ma in age, from borehole cores taken from Death Valley, Saline Valley, and Searles Lake, California, for the first time with laser Raman spectroscopy. Carotenoids occurred in fluid inclusions as colorless to red-brown amorphous and crystalline masses associated with spheroidal algal cells similar in appearance to the common halophilic alga Dunaliella. Spectra from carotenoid standards, including β-carotene, lycopene, and lutein, were compared to microscopically targeted carotenoids in fluid inclusions. Carotenoids produced characteristic bands in the Raman spectrum, 1000-1020 cm⁻¹ (v₃), 1150-1170 cm⁻¹ (v₂), and 1500-1550 cm⁻¹ (v₁), when exposed to visible laser excitation. Laser Raman analyses confirmed the presence of carotenoids with these characteristic peaks in ancient halite. A number of band sets were repeated at various depths (ages), which suggests the stability of this class of organic molecules. Carotenoids appear well preserved in ancient salt, which supports other observations, for example, preserved DNA and live cells, that fluid inclusions in buried halite deposits preserve intact halophilic microbial ecosystems. This work demonstrates the value of laser Raman spectroscopy and carotenoids in extraterrestrial exploration for remnants of microbial life.

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.